Susana R. Valdez

Hormonal profile and reproductive performance in lactation deficient (OFA hr/hr) and normal (Sprague-Dawley) female rats.

Lactation deficiency may have important consequences on infant health, particularly in populations of low socioeconomic status. The OFA hr/hr (OFA) strain of rats, derived from Sprague-Dawley (SD) rats, has deficient lactation and is a good model of lactation failure. We examined the reproductive performance and hormonal profiles in OFA and SD strains to determine the cause(s) of the lactation failure of the OFA strain. We measured hormonal (PRL, GH, gonadotropins, oxytocin, and progesterone) levels by RIA in cycling, pregnant, and lactating rats and in response to suckling. Dopaminergic metabolism was assessed by determination of mediobasal hypothalamic dopamine and dihydroxyphenylacetic acid (DOPAC) concentrations by HPLC and tyrosine hydroxylase expression by immunocytochemistry and western blot. OFA rats have normal fertility but 50% of the litters die of malnutrition on early lactation; only 6% of the mothers show normal lactation. The OFA rats showed lower circulating PRL during lactation, increased hypothalamic dopamine and DOPAC, and impaired milk ejection with decreased PRL and oxytocin response to suckling. Before parturition, PRL release and lactogenesis were normal, but dopaminergic metabolism was altered, suggesting activation of the dopaminergic system in OFA but not in SD rats. The number of arcuate and periventricular neurons expressing tyrosine hydroxylase was higher in SD rats, but hypothalamic expression of TH was higher in OFA rats at the end of pregnancy and early lactation. These results suggest that the OFA rats have impaired PRL release linked with an augmented dopaminergic tone which could be partially responsible for the lactational failure.

Dopaminergic Mechanisms Involved in Prolactin Release after Mifepristone and Naloxone Treatment during Late Pregnancy in the Rat

Background/Aims: During late pregnancy, the antiprogesterone mifepristone facilitates prolactin release. This effect is enhanced by administration of the opioid antagonist naloxone, suggesting an inhibitory-neuromodulatory role of the opioid system. Since hypothalamic dopamine (DA) is the main regulator of prolactin release, in this study we explored the role of DA on prolactin release induced by mifepristone and naloxone treatment. Methods/Results: Rats on day 19 of pregnancy were used. Naloxone treatment did not modify the 3,4-dihydroxyphenylacetic acid/DA (DOPAC/DA) ratio or serum prolactin concentration in control rats. After mifepristone treatment, DA activity diminished significantly without modifying serum prolactin levels. Naloxone administration to antiprogesterone-treated rats did not change the DOPAC/DA ratio but increased serum prolactin. Tyrosine hydroxylase (TH) expression in medial basal hypothalamus (MBH) protein extracts was lowered by pretreatment with mifepristone, with no additional effect of naloxone. While mifepristone decreased the intensity of TH immunoreactivity in the arcuate and periventricular nuclei and in fibers of the median eminence, naloxone treatment had no further effect. Conclusions: (1) A reduction of tuberoinfundibular dopaminergic (TIDA) neuron activity is suggested by the fall of the DOPAC/DA ratio and the low expression of MBH TH; (2) this reduction facilitates prolactin secretion by naloxone, indicating that progesterone stimulates DA neurons to maintain low serum prolactin; (3) naloxone action seems to depend on a previous decrease of DA tone induced by mifepristone, without involve a direct effect on neuronal DA activity, and (4) endogenous opioids may inhibit prolactin secretion through a non-dopaminergic neuronal system that regulates prolactin secretion in which as yet undetermined prolactin-releasing factors may participate.

Advancement of reproductive senescence and changes in the early expression of estrogen, progesterone and µ-opioid receptors induced by neonatal hypoxia in the female rat

Perinatal hypoxia is a frequent birth complication, and although its early consequences on brain development have been well studied, few studies address any long-term effects. Postnatal insults producing small disturbances in endocrine function can have marked and long-lasting effects. In the present work we studied the effects of two types of perinatal brain injury: global hypoxia (H, 6.5% O2 for 50 min) and hypoxia plus ischemia (HI, ligature of the right carotid artery) on female rat reproductive performance and expression of mediobasal hypothalamus-preoptic area (MBH-PO) estrogen, progesterone and micro-opioid receptors at different times after injury, measuring the mRNA (by semiquantitative RT-PCR) and protein (by Western blot). H or HI advanced approximately 3 months after the appearance of blunted preovulatory LH surges and cyclic irregularities (prolonged estrus) characteristic of the early stages of reproductive senescence. 48 h after H or HI we observed decreases in ERbeta, microOR and PR (only in the H group) mRNAs and in total ER and microOR proteins, followed by increased PR levels (mRNA and protein) 7 days post-injury and by increased microOR protein and ERbeta mRNA in the H group and ERalpha, ERbeta and microOR mRNAs and ER protein in the HI group 30 days post-injury. Thus, an episode of hypoxia suffered during early postnatal life induces premature reproductive senescence on the female rats, accompanied by early changes in some MBH-PO hormone receptors (microOR, ER and PR), whose expression is intimately involved in the regulation of gonadotropin secretion and female sexual cyclicity.

Luteal Expression of Thyroid Hormone Receptors During Gestation and Postpartum in the Rat

BACKGROUND Progesterone (P4) is the main steroid secreted by the corpora lutea (CL) and is required for successful implantation and maintenance of pregnancy. Although adequate circulating levels of thyroid hormone (TH) are needed to support formation and maintenance of CL during pregnancy, TH signaling had not been described in this gland. We determined luteal thyroid hormone receptor isoforms (TR) expression and regulation throughout pregnancy and under the influence of thyroid status, and in vitro effects of triiodothyronine (T3) exposure on luteal P4 synthesis. METHODS Euthyroid female Wistar rats were sacrificed by decapitation on gestational day (G) 5, G10, G15, G19, or G21 of pregnancy or on day 2 postpartum (L2). Hyperthyroidism and hypothyroidism were induced in female Wistar rats by daily administration of thyroxine (T4; 0.25 mg/kg subcutaneously) or 6-propyl-2-thiouracil (PTU; 0.1 g/L in drinking water), respectively. Luteal TR expression of mRNA was determined using real-time reverse-transcription quantitative polymerase chain reaction, and of protein using Western blot and immunohistochemistry. Primary cultures of luteal cells and of luteinized granulosa cells were used to study in vitro effects of T3 on P4 synthesis. In addition, the effect of T3 on P4 synthesis under basal conditions and under stimulation with luteinizing hormone (LH), prolactin (PRL), and prostaglandin E2 (PGE2) was evaluated. RESULTS TRα1, TRα2, and TRβ1 mRNA were present in CL, increasing during the first half and decreasing during the second half of pregnancy. At the protein level, TRβ1 was abundantly expressed during gestation reaching a peak at G19 and decreasing afterwards. TRα1 was barely expressed during early gestation, peaked at G19, and diminished thereafter. Expression of TRβ1 and TRα1 at the protein and mRNA level were not influenced by thyroid status. T3 neither modified P4 secretion from CL of pregnancy nor its synthesis in luteinized granulosa cells in culture. CONCLUSIONS This study confirms for the first time the presence of TR isoforms in the CL during pregnancy and postpartum, identifying this gland as a TH target during gestation. TR expression is modulated in this tissue in accordance with the regulation of P4 metabolism, and the abrupt peripartum changes suggest a role of TH during luteolysis. However, TH actions on the CL do not seem to be related to a direct regulation of P4 synthesis.

Alteration of syncytiotrophoblast mitochondria function and endothelial nitric oxide synthase expression in the placenta of rural residents.

The impact of environmental organophosphate (OP) pesticide exposure on respiratory complexes, enzymatic antioxidant defense activities, and oxidative damage markers in the syncytiotrophoblast and cytotrophoblast mitochondria was evaluated. Placental progesterone (PG) levels and endothelial nitric oxide synthase (eNOS) expression were studied. Samples from women non-exposed (control group-CG) and women living in a rural area (rural group-RG) were collected during pesticide spraying season (RG-SS) and non-spraying season (RG-NSS). In RG-SS, the exposure biomarker placental carboxylesterase decreased and syncytiotrophoblast cytochrome c oxidase activity increased, while 4-hydroxynonenal levels decreased. PG levels decreased in RG-SS and in the RG. Nitric oxide synthase expression decreased in RG, RG-SS and RG-NSS. No significant changes in mitochondrial antioxidant enzyme activities were found. These results suggest that the alteration of syncytiotrophoblast mitochondrial complex IV activity and steroidogenic function may be associated to pesticide exposure. Reduction in placental PG and eNOS expression may account for low newborn weight in RG.

Lactation deficit in OFA hr/hr rats may be caused by differential sensitivity to stress compared with Wistar and Sprague Dawley rats

OFA hr/hr (OFA) rats present a major lactation deficit that impairs offspring survival. To explore whether abnormal stress responsiveness causes this deficit, we compared their hormonal (prolactin, progesterone, and corticosterone) responses to stress (room change and 2-min ether exposure) with those of Wistar and Sprague Dawley (SD) rats. We tested responses during the estrous cycle, pregnancy, lactation, after ovariectomy, and ovarian steroid hormone priming, and responses to suckling. We evaluated hypothalamic expression of receptors for prolactin (PRLRlong) and the isoforms of receptors for progesterone (PRA and B) and estrogen (ERα and β) in late pregnancy. We tested whether administration of an anxiolytic (diazepam) improved lactation. Ether exposure increased circulating levels of the three hormones in the three strains of rats, cycling and ovariectomized, but was less effective in pregnancy and lactation. Elevated estrogen level (estrus and estradiol-treated ovariectomized rats) potentiated the prolactin response more in SD and OFA rats than in Wistar rats. Elevated progesterone level (late pregnancy, lactation, progesterone-treated ovariectomized rats) inhibited the prolactin response less in OFA than in SD or Wistar rats. Ether exposure inhibited the prolactin and oxytocin responses to suckling only in OFA rats. Diazepam treatment increased pup survival rate and the prolactin response to suckling. Hypothalamic total PR mRNA content, assayed by RT-PCR, was higher in pregnant OFA rats compared with SD and Wistar rats, but the PRB/PRA protein ratio determined by Western blot was lowest in Wistar rats, intermediate in OFA rats, and highest in SD rats. The heightened sensitivity to stress of lactating OFA rats may contribute to their lactational deficit and be caused by a combination of hypoprolactinemia and reduced inhibitory capacity of progesterone.

Effect of Progesterone Withdrawal on Hypothalamic Mechanisms Related to Prolactin Release in Late Pregnant Rats

Background/Aims: Progesterone (P4) fall provoked by spontaneous or prostaglandin F2α (PGF2α)-induced luteolysis in late pregnant rats triggers a prolactin (PRL) surge 12–24 h later. Methods: To investigate the hypothalamic mechanism mediating this response, we determined expression of tyrosine hydroxylase (TH), PRL receptors (long form, PRLRlong), estrogen-α (ERα) and ERβ, P4 (PR) A and B receptors, and STAT5a, STAT5b, suppressors of cytokine signaling 1 (SOCS1), SOCS3 and CIS at mRNA (by semiquantitative and real-time RT-PCR) and protein (by Western blot only for TH, ERα and PRs) levels, and dopamine and DOPAC (by high-performance liquid chromatography) contents in the mediobasal hypothalamus (MBH) 24 h after luteolysis induced by a PGF2α analogue (cloprostenol, 25 µg/rat s.c. at 8 and 12 h on day 19 of pregnancy). Results: PGF2α treatment decreased circulating P4 and estradiol and increased PRL and the estradiol/P4 ratio. MBH DOPAC and DOPAC/dopamine ratio fell, indicating decreased dopaminergic transmission. PRLRlong, PRB and ERα mRNA increased. ERα and PR proteins were not modified. However, TH protein and mRNA did not change. PRA, the small PR isoform, was much more abundant than PRB, the isoform considered to mediate P4 genomic actions. STAT5a, SOCS1 and SOCS3 mRNA were also increased. Conclusion: The P4 fall induced by PGF2α treatment induces PRL release through diminution in MBH dopaminergic transmission without change in TH expression. The increased PRLR along with elevated circulating PRL may be responsible for maintaining high TH expression through activation of short-loop feedback mechanisms, counteracting the effect of the fall in circulating P4. In parallel, SOCS expression contributes to limit PRL signaling.

Impaired mammary gland T cell population during early lactation in hypoprolactinemic lactation-deficient rats

Mammary stroma is composed of various cell types, including migratory leukocytes. Although mammary antibody-secreting cells have been extensively studied, reports focusing on mammary T cells are scarce. It is thought that the recruitment mechanism of leukocytes to the mammary gland (MG) is controlled by pregnancy- and lactation-specific stimuli. But whether prolactin (PRL) modulates the T-cell population in MG is still unknown. Our aim was to study the relationship between PRL levels and T and B cells during early lactation (L2, day 2 post partum) and mid-lactation (L12, day 12 of lactation). In order to investigate whether PRL is associated with homing events to MG, female Sprague Dawley (SD) and SD-derived desmoglein 4(-/-) hairless (phenotype with lactation deficit, OFA hr/hr) rats were killed during estrus, pregnancy, and post partum, and blood, MG, and corpora lutea were obtained to perform fluorescent-activated cell sorting (FACS), real-time PCR, and histological and RIA studies. Serum PRL levels were lower in OFA hr/hr rats than in SD rats during early lactation. MG of OFA hr/hr rats showed less secretory material compared with SD rats. FACS analysis showed lower percentage of MG CD3+ cells in OFA hr/hr rats compared with SD rats on L2 and L12. OFA hr/hr rats showed higher absolute numbers of circulating CD3+ cells compared with SD rats on L2 but not on L12. These results show that T-cell population in MG is affected in early lactating OFA hr/hr rats and strongly suggest that serum PRL levels may be involved in the homing events to MG, probably helping antibody-secreting cells and protecting the gland during lactation development.

Effect of hyperthyroidism on circulating prolactin and hypothalamic expression of tyrosine hydroxylase, prolactin signaling cascade members and estrogen and progesterone receptors during late pregnancy and lactation in the rat

Hyperthyroidism (HyperT) compromises pregnancy and lactation, hindering suckling-induced PRL release. We studied the effect of HyperT on hypothalamic mRNA (RT-qPCR) and protein (Western blot) expression of tyrosine hydroxylase (TH), PRL receptor (PRLR) and signaling pathway members, estrogen-α (ERα) and progesterone (PR) receptors on late pregnancy (days G19, 20 and 21) and early lactation (L2) in rats. HyperT advanced pre-partum PRL release, reduced circulating PRL on L2 and increased TH mRNA (G21 and L2), p-TH, PRLR mRNA, STAT5 protein (G19 and L2), PRLR protein (G21) and CIS protein (G19). PRs mRNAs and protein decreased on G19 but afterwards PRA mRNA (G20), PRB mRNA (G21) and PRA mRNA and protein (L2) increased. ERα protein increased on G19 and decreased on G20. Thus, the altered hypothalamic PRLR, STAT5, PR and ERα expression in hyperthyroid rats may induce elevated TH expression and activation, that consequently, elevate dopaminergic tone during lactation, blunting suckling-induced PRL release and litter growth.

Abstracts of the 13th Annual ENETS Conference for the Diagnosis and Treatment of Neuroendocrine Tumor Disease. March 9-11, 2016, Barcelona, Spain: Abstracts

ACTG2 Inhibits Growth and Is Epigenetically Repressed in Small Intestinal Neuroendocrine Tumors