Pituitary LH Content Research Articles

OR24-3 Persistence of Progenitor Cell Markers Following the Selective Ablation of Musashi in Somatotropes

Metabolic and reproductive demands are met and coordinated through the complex control of hormone synthesis and secretion exerted by the anterior pituitary. While pituitary cells are known to possess remarkable plasticity to change their cell fate and alter hormone production in response to ever changing environmental cues, the underlying molecular control of this plasticity has not been established. Our lab has previously introduced the Musashi (MSI) family of RNA binding proteins as important players in control of pituitary hormone levels. Musashi typically governs stem cell fate and promotes self-renewal by repressing translation of target mRNAs needed for differentiation. However, we found that MSI1 is expressed in most differentiated cells of the adult pituitary and can specifically bind to the 3’ UTRs of Prl, Tsh, and Pou1f1 and exert translational control in reporter assays. Confirmation of the requirement for MSI was demonstrated through in vivo analyses where MSI1 and MSI2 were selectively ablated in somatotropes. The mutant animals were subfertile. The mutant males showed reduced serum and pituitary content of LH and FSH, and significant decreases in serum GH and PRL despite 2-fold increases in pituitary protein content of PRL and GH. To further assess the role and downstream pathways regulated by MSI in the pituitary, we collected somatotrope MSI-null pituitaries from males and females for qPCR analysis of common somatotrope target genes. In correlation with our previous findings described above (low serum GH and high pituitary GH content), we found that GHRHR mRNA levels were reduced by 2-fold in male mutants. RNA-seq analysis followed by qPCR validation shows that Prop1 mRNA was significantly increased in male mutants, with no significant change in Pou1f1 mRNA. These data suggest that MSI may be involved in the regulation of progenitor cells giving rise to the somatotrope lineage and that MSI ablation may have caused retention of these progenitors and/or a failure to fully differentiate somatotropes. Our studies of MSI-null somatotrope function support this interpretation because the mutant somatotropes clearly stored GH proteins, but could not secrete them, as demonstrated by the low serum GH values, and the 50% reduction in GHRHR mRNA. RNA-seq evaluation of males revealed a change in the expression of 720 genes between controls and mutants (FDR <0.05). When we examined female MSI-null somatotrope animals, a much smaller cohort of genes showed a change in expression (153 genes, FDR <0.05). Interestingly, 38 genes were altered in both mutant males and females suggesting shared regulation by MSI. Further characterization of the NGS datasets will elucidate additional downstream targets and effector pathways of MSI-dependent control of anterior pituitary cell differentiation and function.

Endocrine and reproductive responses of male and female cattle to agonists of gonadotrophin-releasing hormone

The pituitary response in cattle to treatment with GnRH agonist has two phases. In the acute phase secretion of LH is increased, while the chronic phase is characterized by a downregulation of GnRH receptors and insensitivity of gonadotrophs to natural sequence GnRH. After long-term treatment with GnRH agonist, cattle do not have pulsatile secretion of LH but maintain basal LH. This is associated with reduced pituitary contents of LH, LH mRNA, FSH and FSH mRNA. Long-term treatment of bulls with GnRH agonist results in an increase in testicular LH receptors and high plasma testosterone. Heifers treated with a GnRH agonist from early in the oestrous cycle develop a larger corpus luteum and secrete more progesterone. Increased steroidogenesis is reflected in increased steroid acute regulatory (StAR) protein and steroidogenic enzymes in the testes and corpus luteum. GnRH agonists have potential as novel strategies for reproductive management in cattle. A GnRH agonist bioimplant was recently used to block the LH surge after FSH stimulation of follicle growth in heifers. Ovulation was induced by injection of LH, and heifers were inseminated relative to the LH injection. This GnRH agonist-LH protocol provides a model for studying the gonadotrophin requirements for follicular growth and oocyte maturation in cattle, and will enable controlled in vivo maturation of oocytes before recovery for in vitro procedures.

Effects of Short Term Administration of Genistein on Hypothalamic and Anterior Pituitary Hormones in Ovariectomized Gilts

Administration of genistein to barrows increased anterior pituitary (AP) concentrations of IGF-I and LH and increased expression of AP IGF receptor. Whether similar changes occur in gilts remains to be determined. The objective of this experiment was to determine if short term administration of genistein increased expression of components of the AP IGF system and hypothalamic hormones and receptors involved in gonadotropin synthesis and/or release in the gilt. Sixteen crossbred gilts of similar weight (97.7 kg) were ovariectomized and assigned to either control (C; n = 8) or genistein (G; n = 8) groups. Genistein pigs received 800 mg of genistein in DMSO while C pigs received an equal volume of DMSO i.m. on day 0, 1, 2, and 3. Blood samples were obtained on day 0, 1, 2, and 3. Pigs were slaughtered on d 4 when blood, AP, and medial basal hypothalami (MBH) were collected. No difference was detected (P > 0.05) in AP concentrations of IGF-I or serum concentrations of IGF-I in C and G pigs. Anterior pituitary concentrations of LH were greater (P 0.05) in C and G pigs. Relative expression of AP IGFBP-5 and GnRHR was increased (P < 0.05) in G pigs compared with C pigs. Relative expression of AP LHβ did not differ between C and G pigs. Relative expression of MBH kisspeptin was greater (P < 0.01) in G pigs than C pigs. These data provided evidence that short term administration of genistein increased expression of hypothalamic and hypophyseal hormones in gilts which could influence subsequent reproduction.

Pituitary gonadotrophic hormone synthesis, secretion, subunit gene expression and cell structure in normal and follicle-stimulating hormone β knockout, follicle-stimulating hormone receptor knockout, luteinising hormone receptor knockout, hypogonadal and ovariectomised female mice.

To investigate the relationship between gonadotroph function and ultrastructure, we have compared, in parallel in female mice, the effects of several different mutations that perturb the hypothalamic-pituitary-gonadal axis. Specifically, serum and pituitary gonadotrophin concentrations, gonadotrophin gene expression, gonadotroph structure and number were measured. Follicle-stimulating hormone β knockout (FSHβKO), follicle-stimulating hormone receptor knockout (FSHRKO), luteinising hormone receptor knockout (LuRKO), hypogonadal (hpg) and ovariectomised mice were compared with control wild-type or heterozygote female mice. Serum levels of LH were elevated in FSHβKO and FSHRKO compared to heterozygote females, reflecting the likely decreased oestrogen production in KO females, as demonstrated by the threadlike uteri and acyclicity. As expected, there was no detectable FSH in the serum or pituitary and an absence of expression of the FSHβ subunit gene in FSHβKO mice. However, there was a significant increase in expression of the FSHβ and LHβ subunit genes in FSHRKO female mice. The morphology of FSHβKO and FSHRKO gonadotrophs was not significantly different from the control, except that secretory granules in FSHRKO gonadotrophs were larger in diameter. In LuRKO and ovariectomised mice, stimulation of LHβ and FSHβ mRNA, as well as serum protein concentrations, were reflected in subcellular changes in gonadotroph morphology, including more dilated rough endoplasmic reticula and fewer, larger secretory granules. In the gonadotophin-releasing hormone deficient hpg mouse, gonadotrophin mRNA and protein levels were significantly lower than in control mice and gonadotrophs were correspondingly smaller with less abundant endoplasmic reticula and reduced numbers of secretory granules. In summary, major differences in pituitary content and serum concentrations of the gonadotrophins LH and FSH were found between control and mutant female mice. These changes were associated with changes in expression of the gonadotrophin subunit genes and were reflected in the cellular structure and secretory granule appearance within the gonadotroph cells.

Effects of Sulfamethazine on Induction of Precocious Puberty in Japanese Quails (&lt;i&gt;Coturnix japonica&lt;/i&gt;) Assessed through Monitoring the Hormonal Changes and Gonadal Development

To investigate the effect of dietary supplementation of sulfamethazine (SMZ) on growth performance, gonadal development and hormonal changes, male and female Japanese quails (Coturnix japonica) were fed a control diet with or without SMZ (0.2%) from one day post hatching until 6 weeks of age. In male quail, the deviation in growth performance between SMZ and control chicks started at the 3rd week, and the disparity was significant at the 5th and 6th weeks. Hormonal analysis revealed a substantial increase in the pituitary and circulating LH (at the 5th and 6th weeks), testicular and circulating testosterone (at the 6th week) and plasma ir-inhibin (at 5th week) levels following feeding of the diet containing SMZ. The testicular size and weights were significantly larger at the 5th week, and histological analysis demonstrated an enlargement of seminiferous tubules, filling of the luminal fluid with spermatozoa and a number of interstitial cells. In female quail, the body and ovarian weights were considerably increased at the 6th week. The SMZ supplemented group showed a significant elevation in pituitary LH content (from the 4th week), plasma LH (at the 5th and 6th weeks), ir-inhibin (at the 3rd and 6th week) and progesterone (at the 2nd, 5th and 6th weeks) as compared with control chicks. These results indicated that SMZ was able to stimulate the secretion of gonadotropins and accordingly the gonadal hormones and that was associated with an early gonadal function in male (at the 5th week) and female (at the 6th week) Japanese quail.

Effects of immunization against luteinizing hormone-releasing hormone and treatment with trenbolone acetate on reproductive function of beef bulls and steers1

The objectives of this study were 1) to evaluate the ability of trenbolone acetate (TBA) administered in tandem with LHRH immunization to suppress reproductive function in bulls and 2) to examine the effects of LHRH and androgen (TBA) signaling on pituitary gland function. Forty-four Angus × Hereford crossbred calves (BW=225 ± 2 kg; age=187 ± 6 d) received castration, LHRH immunization, or TBA administration in a 2 × 2 × 2 factorial design. Treatment groups receiving LHRH immunization contained 6 animals, whereas other treatment groups contained 5 animals. Animals immunized against LHRH received a primary injection and 2 booster injections of ovalbumin-LHRH-7 fusion protein on d 0, 42, and 196, respectively. Animals treated with TBA were implanted on d 224. Serum LHRH antibodies increased (P<0.05) after each booster for immunized animals, but were negligible in nonimmunized animals throughout the experiment. Serum testosterone concentration (P<0.001) and scrotal circumference (P<0.05) were depressed in LHRH-immunized bulls compared with nonimmunized bulls by d 84 and 168 of the experiment, respectively. Treatment with TBA tended (P=0.08) to decrease serum testosterone concentrations of nonimmunized bulls. Weights of testes at slaughter were decreased (P<0.001) for LHRH-immunized (232 ± 41 g) compared with nonimmunized (752 ± 45 g) bulls, but did not differ (P=0.80) between TBA-implanted (500 ± 49 g) and nonimplanted bulls (484 ± 36 g). Both LHRH immunization and castration decreased pituitary gland stores of LH and FSH (P<0. 001). There was no effect (P>0.10) of TBA on pituitary gland FSH content and only a tendency (P=0.09) to increase pituitary gland LH content. Immunization against LHRH decreased expression of LH β-subunit and common α-subunit genes (P<0.001). Castration increased expression of LH β-subunit and common α-subunit genes (P=0.02). Treatment with TBA further suppressed (P=0.04) α-subunit mRNA expression in LHRH-immunized steers. In summary, LHRH immunization decreased synthesis and storage of LH and decreased storage, but not synthesis of FSH in bulls. The increased synthesis of LH and FSH in nonimmunized, but not LHRH-immunized steers suggests that castration removes the negative feedback on gonadotropin synthesis but that LHRH is still needed for release of these hormones. Androgen replacement with TBA did not restore the negative feedback control of gonadotropin synthesis.

Involvement of rat gonadotrope progesterone receptor in the ovary-mediated inhibitory action of FSH on LH synthesis

Rat ovaries stimulated with human follicle-stimulating hormone (hFSH) overexpress a factor that attenuates the LH surge in the rat: the putative gonadotropin surge-attenuating factor (GnSAF). A reduced gondadotrope progesterone receptor (PR) phosphorylation/activation is likely to be the main causative factor involved in GnSAF bioactivity on LH release. Besides, GnSAF reduces LH synthesis as well as LH secretion, and it is not known whether PR is involved in the inhibitory action of GnSAF on LH synthesis. Thus, the purpose of the present work was to evaluate the involvement of PR in the inhibitory effects of GnSAF on LH synthesis in cycling rats. To this end we used a specific radioimmunoassay and reverse transcription-polymerase chain reaction (RT-PCR) to study the effect on LH pituitary content and LHβ mRNA expression of PR occupancy with P (3 mg/0.2 ml oil in diestrus) on the inhibitory effects of hFSH (0, 0.1, 1, and 10 IU) in metestrus (day 2) and diestrus (day 3) on LH synthesis on proestrus in intact and on day 4 in day 2 ovariectomized (OVX) rats injected with 5 and 10 μg of estradiol benzoate (EB) on days 2 and 3, respectively. Results showed that (1) hFSH decreased pituitary LH content in intact, but not in OVX rats injected with EB, without affecting LHβ mRNA levels, and (2) PR occupancy with P annulled the inhibitory action of hFSH on pituitary LH content. These results indicate that PR is involved in ovarian GnSAF effect on LH content probably at a post-transcriptional level.

Plasma and pituitary gonadotropins and prolactin after hysterectomy and treatment with uterine extracts in female rats. A kinetic study.

The kinetic of changes in plasma concentrations and pituitary contents of FSH, LH, prolactin after hysterectomy and treatment with steroid-free uterine extracts were studied in adult, non-pregnant female rats. The post-castration rise of plasma and pituitary FSH and plasma LH was inhibited but the plasma prolactin was elevated by hysterectomy. Steroid-free uterine extracts from previously castrated animals increased the plasma FSH, LH and prolactin concentrations in hysterectomized females 48 hours after administration. Uterine extracts from intact and oestradiol-substituted-castrated animals decreased the pituitary prolactin content 48 h after single i.p. injection. Pituitary FSH and LH contents were not affected by single dose of uterine extracts at any time which were examined. It is concluded that the uterus may play some role in the synthesis and release of pituitary gonadotropins and prolactin.

Effects of hysterectomy and uterine extracts on the gonadotrophic hormones and the weight of endocrine organs of female rats.

The previous observation that hysterectomy of rats results in increased pituitary nucleic acid turnover was followed up by studies on pituitary function in hysterectomized adult female rats with or without treatment with charcoal-treated crude uterine aqueous extracts. Hysterectomy had no effect on the pituitary or plasma LH and FSH concentrations. Administration of crude uterine extracts diminished the post-castrational rise of the pituitary FSH content without affecting the plasma FSH or LH concentrations. Hysterectomy decreased body weight but increased adrenal weight of ovariectomized animals. However, the body weight and pituitary weight of both the intact and ovariectomized animals increased significantly following 14 days of treatment with uterine extracts. The ovarian weight of the intact rats was reduced by this treatment. The pituitary nucleic acid content of castrated but oestradiol-substituted rats was reduced by hysterectomy. Plasma corticosterone concentrations were not affected by the above mentioned treatments. Uterine extracts inhibited the basal and GnRH induced LH release from isolated pituitary cells in culture. The effects of hysterectomy and/or administration of crude uterine aqueous extracts show that the uterus contains biologically active, non-steroidal substances which affect pituitary function of adult female rats.

Effect of estradiol benzoate on the pituitary-gonadal axis in the intact male rat.

Adult male rats were treated with 50 microgram of estradiol benzoate daily for 28 days. The sex organs and pituitary weights showed changes classically associated with estrogen treatment. Both plasma and testicular testosterone levels diminished to almost trace level 24 hours after the first estrogen injection and remained at that level throughout the experimental period. The pituitary LH levels and concentration showed a progressive decline; however, the plasma LH levels remained unchanged throught the experimental period. These findings suggest that in intact adult males, estrogen affects testicular testosterone production directly. These observation suggests the necessity for reevaluation of the current concepts concerned with testis-pituitary feedback mechanisms.

Estrogen Receptor α Signaling Pathways Differentially Regulate Gonadotropin Subunit Gene Expression and Serum Follicle-Stimulating Hormone in the Female Mouse

Estrogen, acting via estrogen receptor (ER)alpha, regulates serum gonadotropin levels and pituitary gonadotropin subunit expression. However, the cellular pathways mediating this regulation are unknown. ERalpha signals through classical estrogen response element (ERE)-dependent genomic as well as nonclassical ERE-independent genomic and nongenomic pathways. Using targeted mutagenesis in mice to disrupt ERalpha DNA binding activity, we previously demonstrated that ERE-independent signaling is sufficient to suppress serum LH levels. In this study, we examined the relative roles of ERE-dependent and -independent estrogen signaling in estrogen regulation of LH, FSH, prolactin, and activin/inhibin subunit gene expression, pituitary LH and FSH protein content, and serum FSH levels. ERE-independent signaling was not sufficient for estrogen to induce pituitary prolactin mRNA or suppress pituitary LHbeta mRNA, LH content, or serum FSH in estrogen-treated ovariectomized mice. However, ERE-independent signaling was sufficient to reduce pituitary glycoprotein hormone alpha-subunit, FSHbeta, and activin-betaB mRNA expression. Together with previous serum LH results, these findings suggest ERE-independent ERalpha signaling suppresses serum LH via reduced secretion, not synthesis. Additionally, ERE-dependent and ERE-independent ERalpha pathways may distinctly regulate steps involved in the synthesis and secretion of FSH.

High prevalence rate of pituitary incidentaloma: Is it associated with the age-related decline of the sex hormones levels?

Incidental pituitary adenoma is the common finding during brain imaging. According to multistep model of pituitary tumourigenesis genetic alterations provide the initiating event that transforms cells while hormones play a role in promoting cell proliferation. Development of pituitary adenoma in a case of excessive hypophysiotrophic hormones production or reduced feedback suppression by target gland hormones emphasizes the importance of hormonal stimulation in pituitary tumourigenesis. Pituitary hyperplasia has been reported in pregnancy, hypothyroidism and conditions such as CRH or GHRH hypersecretion. Moreover, recent study reported one case of gonadotroph macroadenoma and two cases of gonadotroph cells hyperplasia in patients with Klinefelter syndrome probably due to protracted stimulation of gonadotroph cells because of lack of androgen feedback. Significant changes of the hypothalamic-pituitary-gonadal axis occurred with aging. In females, after menopause, estradiol level decreases by 35-fold and estrone level by 20-fold that results in increased gonadotropins levels. Similarly, FSH, but not LH, level is increased with advancing age in men, too, although the age-related difference in the level is less in comparison with women. Regarding these data, we hypothesised that high prevalence rate of pituitary incidentaloma in the elderly is associated with age-related decline in sex hormones levels and subsequent lack of feedback suppression leading to permanent gonadotrophs stimulation which is the crucial step in the pituitary tumour development. According to previously mentioned multistep model of pituitary tumourigenesis, incidentaloma will develop only in persons with already present intrinsic pituitary cell defects. However, further studies have to answer the questions of whether the incidence of pituitary tumours is more frequent in elderly, whether women with late onset menopause or those taking long-term hormone replacement therapy have lower rate of pituitary incidentaloma, and finally, is there any correlation between pituitary tumours incidence and serum concentrations of LH, FSH, bioavailable testosterone or estradiol.

Influence of short-term fasting during the luteal phase of the estrous cycle on ovarian follicular development during the ensuing proestrus of ewes

Short-term fasting of mature ewes during diestrus results in increased serum concentrations of progesterone and a delayed pre-ovulatory surge release of LH. To determine if these changes in reproductive hormones influence subsequent follicular development, mature ewes observed in estrus were assigned randomly to control ( n = 10) or fasted ( n = 15) groups. Control ewes had ad libitium access to feed, whereas fasted ewes were not fed from day 7 through 11 of their estrous cycle. Daily blood samples were collected from control and fasted ewes throughout the fasting period. Fasting increased ( P < 0.001) serum concentrations of progesterone (4.4 ng/mL versus 2.7 ng/mL [±0.3]). On day 12, all ewes were treated with 10 mg of PGF 2α and fasted ewes were returned to ad libitum feed. Ovaries were collected from ewes ( n = 5 each group) at 0 and 72 h following PGF 2α in control and 0, 72 and 96 h in fasted ewes. Ovaries were weighed and small (≤2 mm), medium (3–4 mm), and large (≥5 mm) follicles were enumerated. Total numbers of follicles were less ( P < 0.001) in fasted than fed ewes (14.6 versus 30.2 [±2.2]) at 0 h, but did not differ ( P = 0.9) when numbers of follicles were compared at similar times before the anticipated LH surge (i.e., at 72 h versus 96 h in control and fasted ewes, respectively). Within follicular size class, numbers of small and medium follicles were decreased ( P = 0.04) at 0 h in fasted ewes. Numbers of large follicles did not differ ( P = 1.0) between groups. Although numbers of small and medium ovarian follicles in fasted ewes recovered by 96 h to values comparable to fed ewes at 72 h following PGF 2α, serum concentrations of estradiol 17β ( P = 0.08) and FSH ( P = 0.06) tended to be decreased in fasted ewes before the anticipated surge release of LH. Pituitary content of LH and FSH also tended to be lower ( P ≤ 0.09) at 96 h in fasted ewes than at 72 h in control ewes, but did not differ ( P ≥ 0.4) at hour 0 following PGF 2α. Hypothalamic and stalk median eminence contents of GnRH were not influenced ( P ≥ 0.2) by fasting at any time period. Fasting during the luteal phase perturbs gonadotropin secretion and may influence fertility by causing a delay in ovarian follicle development.

Gonadotropin-secreting cells in ovariectomized rats treated with different oestrogen receptor ligands: a modulatory role for ERβ in the gonadotrope?

In the rat, oestrogen is a key regulator of gonadotrophin synthesis and release through activation of oestrogen receptors (ERs). Gonadotropes express alpha and beta isoforms of ER and both can activate transcription in response to oestrogen. These experiments were aimed at evaluating the relative contribution of ERalpha and ERbeta on gonadotrope morphology, progesterone receptor (PR) expression and LH secretion. Ovariectomized rats were daily injected over 3 days with 25 microg oestradiol benzoate, 0.3 or 1.5 mg of the selective ERalpha agonist propylpyrazole triol (PPT) with or without 1.5, 3.0 or 4.5 mg of the selective ERbeta agonist diarylpropionitrile (DPN), DPN alone, and 0.3 or 3 mg of tamoxifen. Controls were given 0.2 ml oil. Serum concentration and pituitary content of LH, gonadotrope PR expression, pituitary PR content, and gonadotrope morphology were analyzed by RIA, immunohistochemistry, Western blotting and light and electron microscopy, respectively. Results showed that PPT reversed all consequences of ovariectomy, DPN mimicked the effects of PPT except for its LH-releasing action and tamoxifen had ERalpha-like responses. When combined with PPT, DPN attenuated ERalpha effects without interfering with its LH-releasing activity. Oestradiol benzoate had similar effects to those of combined PPT and DPN. It is suggested that (i) the structural reorganization of the cytoplasmic organelles provided by oestrogen, and the shrinkage of the ovariectomy-induced hypertrophy of gonadotropes, which precedes the expression of PR, are evoked by ERalpha and modulated, in a ying-yang fashion, by ERbeta; and (ii) the oestrogen-dependent exocytosis of LH, the final step in the secretory process, is dependent on ERalpha exclusively.

Estrogenic induction of spermatogenesis in the hypogonadal (hpg) mouse: role of androgens

Testicular development is arrested in the hypogonadal (hpg) mouse due to a congenital deficiency of hypothalamic gonadotropin-releasing hormone synthesis. Previous studies have demonstrated that chronic treatment of these mice with estradiol induces testicular maturation and qualitatively normal spermatogenesis, but it is not known whether these are direct effects via estrogen receptors expressed in the testis, or indirect actions via the pituitary gland. The aim of the current studies was to determine whether the actions of estradiol require the presence of androgens. Sensitive assays revealed that chronic estradiol treatment produced time-dependent increases in pituitary FSH production but no increases in pituitary LH or testicular testosterone content could be detected. As a functional test of androgen dependence, hpg mice were treated for 70 days with estradiol plus Casodex (bicalutamide), an androgen receptor antagonist. Casodex treatment markedly attenuated both the estradiol-induced increase in testicular weight and the proliferation of the seminiferous epithelium, as revealed by morphometric analysis. However, it did not affect the estradiol-induced increase in pituitary FSH content, nor did it affect estradiol-induced increases in the weight of the seminal vesicles and epididymides. We conclude that increased FSH production is not sufficient to explain the increase in testicular development induced by estradiol in hpg mice; there is a requirement for functional androgen receptors for induction of testicular growth.

Effect of copper status, supplementation, and source on pituitary responsiveness to exogenous gonadotropin-releasing hormone in ovariectomized beef cows1,2,3

The effect of Cu status, supplementation, and source on pituitary responsiveness to exogenous GnRH was evaluated using nine multiparous, nonpregnant, nonsuckling, ovariectomized Angus cows (7.1 +/- 3.3 yr; 622.9 +/- 49.8 kg; BCS = 6.0 +/- 0.5). Cows were considered Cu-deficient based on liver Cu concentrations (< 30 mg of Cu/kg of DM) after receiving a low-Cu, forage-based diet supplemented (DM basis) with 5 mg of Mo/kg and 0.3% S for 216 d. Copper-deficient cows were stratified based on age, BW, BCS, and liver Cu concentration and assigned randomly to repletion-phase treatments. Treatments included 1) control (no supplemental Cu); 2) organic (ORG; 100% organic Cu); and 3) inorganic (ING; 100% inorganic CuSO4). Treatments were formulated to meet all NRC recommendations, except for Cu, which was supplemented to ORG and ING cows at 10 mg of Cu/kg of dietary DM. During the 159-d repletion phase, Cu status was monitored via liver biopsy samples, and all cows received exogenous progesterone. A controlled intravaginal drug-release device (replaced every 14 d) was used to maintain luteal phase progesterone as a means to provide negative feedback on the hypothalamic-pituitary axis. During the repletion phase, liver Cu concentrations did not differ between ORG and ING cows at any time. By d 77 of the repletion phase, all supplemented cows were considered adequate in Cu, and liver Cu concentrations were greater in supplemented than in nonsupplemented control cows on d 77 (P < 0.05) and throughout (P < 0.01) the repletion phase. Beginning on d 99, exogenous GnRH was administered to all cows at low (0, 3, and 9 microg; Exp. 1) and high doses (0, 27, and 81 microg; Exp. 2) at six different times. Cows were catheterized every fifth day, and blood samples were collected every 15 min for 1 h before and 4 h after GnRH administration and analyzed for LH concentration. In Exp. 1, Cu status and supplementation did not affect basal or peak LH concentrations, but total LH released tended (P < 0.07) to be greater in Cu-supplemented vs. control cows when 3 microg of GnRH was administered. In Exp. 2, there was no effect of Cu supplementation or source on basal, peak, or total LH released, regardless of GnRH dose. Pituitary LH concentrations did not differ across treatments. In conclusion, Cu status, supplementation, and source did not affect GnRH-induced LH secretion or pituitary LH stores in ovariectomized, progesterone-supplemented cows in this experiment.

Atrial natriuretic peptide inhibits ovarian functions in female mice

Objective: To investigate the effects of atrial natriuretic peptide (ANP) on ovarian regulation in mice through intraperitoneal administration. Study design: Forty-two ICR strain female mice were divided into seven groups, including one control. Each mouse received ovarian hyperstimulation. ANP was prepared with saline at concentrations of 0 (as control), 2, 4, 8, 10, 20, and 30 ng/g of body weight. The solution was intraperitoneally applied to each referred group ( n = 6) 1 h before the hMG and hCG injections. The mice were sacrificed next morning and blood samples were collected for analysis. Results: A significant reduction in both ovarian weight and the number of oocytes was found as the ANP dosage increased. ANP treatment decreased both serum estradiol and progesterone levels. The pituitary FSH and LH contents elevated significantly by ANP at a dosage larger than 4 ng/g of body weight; however, both serum FSH and LH levels remained unchanged. Histologically, there were remarkable ovarian inhibitory features, i.e. reduction in size and number of follicles, arrest of follicular growth and diminution of ovulation. Conclusion: Intraperitoneal administration of ANP inhibits the follicular growth, ovulation and steroidogenesis in female mice. These effects are attributed to the direct action of ANP on ovaries.

GnRH Antagonist-induced Down-regulation of the mRNA Expression of Pituitary Receptors: Comparisons with GnRH Agonist Effects

In order to compare the mechanism for the down regulation of the mRNA expression of pituitary receptors induced by GnRH antagonist (GnRHant) to that by GnRH agonist (GnRHa), we examined the effects of GnRHant (Cetrorelix, 333 mug/kg/day), GnRHa (leuprolide depot, 333 microg/kg), and GnRHant combined with GnRHa on LH response to exogenous GnRH, pituitary LH content, LH beta subunit mRNA, and GnRH receptor (GnRH-R) mRNA levels at 2, 5, 24, 72 hours, and 7 days after the treatment in ovariectomized rats. GnRHant significantly decreased serum LH, the LH response of the pituitary to exogenous GnRH, and the pituitary LH content compared to the control treatment, though GnRHa significantly increased serum LH. GnRHant with GnRHa significantly diminished the GnRHa-induced flare-up phenomenon. GnRHant significantly decreased LH beta mRNA and GnRH-R mRNA levels, but the magnitude of the decrease in these mRNA levels by GnRHant was significantly less than those by GnRHa until 72 hours following treatment. Prolonged treatment of GnRHant caused a marked inhibition of LH beta mRNA and GnRH-R mRNA expression, similar to that caused by GnRHa. Combination treatment with GnRHa and GnRHant was demonstrated to decrease LH beta mRNA and GnRH-R mRNA levels as much as GnRHa alone and GnRHant alone over 7 days of the treatment. The present study showed differences between GnRHant and GnRHa treatment in the reduction of GnRH-R mRNA levels up to 72 hours after the treatment, and indicated that the suppression of GnRH-R mRNA by GnRHant was the maximal by GnRHa 7 days after the treatment because more profound suppression was not observed upon additional treatment with GnRHa. The findings in the present study support the hypothesis that the mechanism by which GnRHant leads to down-regulation of the mRNA expression of pituitary receptors is similar to that of GnRHa.

Oestradiol-17β responsiveness, plasma LH profiles, pituitary LH and FSH concentrations in long-term ovariectomised Holstein cows at 24 h, 48 h and 21 days following treatment with an absorbable GnRH agonist implant

Non-lactating OVX Holstein cows ( N = 34) were used to investigate the effect of s.c. placement of an absorbable GnRH agonist implant (Ovuplant ®; deslorelin 2.1 mg, Peptech Animal Health, Australia) on the relationship of plasma LH, oestradiol responsiveness and pituitary LH content. On the day of implant insertion (Day 0), one group (OVU-48h; N = 5) received Ovuplant ® and had blood samples collected at hourly intervals to characterize the LH response, while a second group (CON-48h; N = 5) remained untreated and acted as controls. Blood samples were collected every 10 min over 6 h from CON-48h and OVU-48h, at 24 h post-implant insertion. These cows were then slaughtered at 48 h post-implant insertion and their pituitaries recovered. Another group received Ovuplant ® (OVU-21d+E2; N = 10) or were left untreated (CON-21d+E2) and 21 days later were injected i.m. with 0.5 mg 17β-E2. Blood samples were collected every 10 min for 4 h on the day before E2 injection to characterize LH pulse frequency and amplitude. Beginning 14 h later, blood samples were collected hourly for 12 h to characterize the expected LH surge. These cows were slaughtered and their pituitary glands recovered and assayed for LH and FSH content. Peak plasma LH concentrations (59±19 ng/ml) were measured after 30 min of Ovuplant ® insertion. They had returned to pre-treatment levels by 7 h. By 24 h post-implant insertion, OVU-48h plasma LH profiles were characterized by reduced LH pulse frequency (0.23 ± 0.09 pulses/h versus 0.75 ± 0.26 pulses/h; OVU-48h versus CON-48h; P < 0.05). The cows that received Ovuplant ® had lower LH pulse amplitude, LH pulse frequency and mean LH concentrations after 20 days. Injection of 0.5 mg 17β-E2 induced an LH surge in every one of the control cows with their peak concentrations measured 18 h post injection. No increase in LH was detected in any Ovuplant ® treated cows. Pituitary FSH content was reduced in Ovuplant ® treated cows after 48 h, but not that of LH. In conclusion, absorbable deslorelin implants induced a substantial but temporary release of LH, but even 21 days later their LH profiles were characterized by marked suppression of pulsatile LH and an absence of response to E2. These results suggest the implant has prolonged biological activity.

Effect of increasing levels of undegradable intake protein on metabolic and endocrine factors in estrous cycling beef heifers.

To determine the influence of three levels of undegradable intake protein (UIP) supplementation on metabolic and endocrine factors that influence reproduction, 23 yearling crossbred heifers (body condition score = 4.5 +/- 0.5; initial BW = 362 +/- 12 kg) were stratified by BW and assigned randomly to one of three supplements: 1) low UIP (1,135 g x heifer(-1) x d(-1); 30% CP, 115 g UIP, n = 7); 2) mid UIP (1,135 g x heifer(-1) x d(-1); 38% CP, 216 g UIP, n = 8); or 3) high UIP (1,135 g x heifer(-1) x d(-1); 46% CP, 321 g UIP, n = 8). Heifers were estrually synchronized before initiation of supplementation. Supplement was individually fed daily for 30 to 32 d, at which time heifers were slaughtered (d 12 to 14 of the estrous cycle) and tissues collected. Heifers were fed a basal diet of sudan grass hay (6.0% CP) ad libitum. On d 28 of supplementation (d 10 of the estrous cycle), no differences were observed (P > 0.10) in serum insulin or IGF-I among treatments. At slaughter (d 10 to 12 of the estrous cycle), treatments did not influence corpus luteum weight, cerebral spinal fluid leptin, or IGFBP; serum estradiol-17beta, progesterone, leptin, IGF-I, and IGFBP; or anterior pituitary content of IGFBP (P > 0.10). Follicular fluid IGFBP-2 and IGFBP-4 were greater in high-UIP heifers than low- or mid-UIP heifers on d 12 to 14 of the estrous cycle (P < 0.05). Basal serum LH concentrations and LH area under the curve (every 15 min for 240 min) did not differ (P > 0.10) following 28 d of supplementation (d 10 of the estrous cycle); however, basal serum FSH concentrations were greater (P = 0.06) in low- and mid- vs. high-UIP heifers (5.2 and 5.2 vs. 4.6 ng/mL, respectively), and FSH area under the curve was greater (P = 0.03) in low- vs. high-UIP heifers. At slaughter (d 12 to 14 of the estrous cycle), anterior pituitary LH and FSH content and steady-state mRNA encoding alpha, LHbeta, and GnRH receptor did not differ (P > 0.10) among treatments. However, FSHbeta mRNA was increased approximately twofold (P = 0.03) in mid vs. low UIP. In summary, low and mid levels of UIP supplements fed to estrous cycling beef heifers seemed to enhance pituitary expression and/or secretion of FSH relative to high levels of UIP. Moreover, high-UIP supplementation was associated with increased low-molecular-weight IGFBP compared with supplementation of low and mid levels of UIP. These data suggest that differing levels of UIP supplementation may alter pituitary and ovarian function, thereby influencing reproductive performance in beef heifers.