Graciela A. Jahn

Short term hypothyroidism affects ovarian function in the cycling rat

BackgroundRats made hypothyroid with propilthyouracil start showing abnormal cycling on the second cycle after the start of the treatment, with a high proportion of spontaneous pseudopregnancies and reduced fertility.MethodsTo investigate some of the mechanisms involved in these reproductive abnormalities, hypothyroidism was induced in virgin rats by propilthyouracil (0.1 g/L in the drinking water) and we determined circulating hormones by radioimmunoassay and whole ovary expression of ovarian hormone receptors, growth factors and steroidogenic enzymes using semi-quantitative RT-PCR.The study was performed on days 6 to 9 of treatment, corresponding to diestrus I (at 20.00-22.00 h), diestrus II (at 20.00-22.00 h), proestrus and estrus (both at 8.00-10.00 h and 20.00-22.00 h) of the second estrous cycle after beginning propilthyouracil treatment. Another group of rats was mated on day 8 and the treatment continued through the entire pregnancy to evaluate reproductive performance.ResultsHypothyroidism increased circulating prolactin and estradiol on estrus 5 to 7-fold and 1.2 to 1.4-fold respectively. Growth hormone and insulin-like growth factor 1 diminished 60 and 20% respectively on proestrus morning. Hypothyroidism doubled the ovarian mRNA contents of estrogen receptor-beta on proestrus and estrus evenings, cyp19A1 aromatase mRNA on estrus evening and of growth hormone receptor on proestrus evening. Hypothyroidism did not influence ovulation rate or the number of corpora lutea at term, but a diminished number of implantation sites and pups per litter were observed (Hypothyroid: 11.7 +/- 0.8 vs. Control: 13.9 +/- 0.7).ConclusionsShort term hypothyroidism alters normal hormone profile in the cycling rat increasing the expression of estrogen receptor-beta and cyp19A1 aromatase on estrus, which in turn may stimulate estradiol and prolactin secretion, favouring corpus luteum survival and the subsequent instauration of pseudopregnancy.

Characterization of seasonal reproduction patterns in female pichis Zaedyus pichiy (Xenarthra: Dasypodidae) estimated by fecal sex steroid metabolites and ovarian histology

Reproductive strategies vary considerably among species, but most studies have focused on a very limited number of mammalian species. Knowledge of the reproductive cycle and behavior is essential for developing and implementing in situ and ex situ conservation strategies for threatened and endangered species. This study aimed at characterizing the seasonal reproductive pattern of female pichis Zaedyus pichiy, a threatened small armadillo native to arid regions of Argentina and Chile, through direct observations, histological studies, and by measuring fecal immunoreactive estrogens, progestagens and glucocorticoids in 10 wild-born, captive pichis and in free-ranging individuals. Results suggest that pichis are seasonal breeders that give birth to one yearly litter of 1-2 offspring, which do not leave the burrow until they are weaned at approximately 37 days. Ovarian follicular growth seems to occur throughout the year. Fecal progestagen, estrogen and glucocorticoid concentrations were minimal during the first half of pregnancy, increased to peak concentrations of up to 3500, 200 and 200ng/g dry feces, respectively, and decreased before parturition. Postpartum progestagen concentrations were greater in lactating females than females that aborted or did not raise their offspring (p<0.0001), which is probably related to an elevated corticosteroid synthesis that contributes to maintain lactation, given that fecal glucocorticoid concentrations were of similar pattern. Observations of a second pregnancy after late abortion or death of the newborn litter and sustained follicular growth during pregnancy and lactation suggest that female pichis can become receptive briefly after having lost their litter. Fecal estrogen and progestagen concentrations of non-pregnant, non-lactating females did not have a well-defined hormonal cyclic pattern, and corpora lutea were only observed in pregnant females.

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.

Alterations of folliculogenesis in women with polycystic ovary syndrome

The objective of the present study was to examine some factors involved in follicular development of women with polycystic ovary syndrome (PCOS). Women with PCOS showed increased levels of serum luteinizing hormone (LH) but decreased follicular production of progesterone and estradiol by pre-ovulatory follicles. The mRNA expression corresponding to steroidogenic acute regulatory protein (StAR), and 20alpha-hydroxysteroid dehydrogenase (20α-HSD) was increased, while that corresponding to cytochrome P450 aromatase (P450arom) was decreased in PCOS follicles as compared to controls. No changes in the mRNA expression for 3beta-hydroxysteroid dehydrogenase 2 (3β-HSD2), cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 alpha hydroxylase/lyase (P450c17), cyclooxygenase 2 (COX2), and transcription factors (GATA-4 and GATA-6) were found. We conclude that despite the hyper-luteinized environment of PCOS follicles, these follicles produce lower levels of progesterone and estradiol, and that this is characterized by increased degradation of progesterone and decreased estradiol synthesis. Our data demonstrate that the synthesis of prostaglandin F2α (PGF2α) may be affected in PCOS-follicles and that the transcription factors GATA-4 and GATA-6 are present in PCOS-follicles but they are not involved in the abnormal transcription observed in the steroidogenic enzymes.

Estrogen inhibits tuberoinfundibular dopaminergic neurons but does not cause irreversible damage

Dopaminergic neurons of the hypothalamic tuberoinfundibular dopaminergic (TIDA) system exert a tonic inhibitory control on prolactin (PRL) secretion whereas estrogen, known to inhibit TIDA neuron function, has been postulated to be toxic to TIDA neurons when it is chronically high. In order to determine whether estrogen in high doses can cause permanent damage to TIDA function, we submitted young female rats to continue high doses of estrogen administered, either centrally (intrahypothalamic estrogen implants) or peripherally (subcutaneous estrogen implants or weekly intramuscular (i.m.) injections for 7 weeks), subsequently withdrawing the steroid and observing the evolution of lactotrophes, serum PRL and TIDA neurons. Serum PRL was measured by radioimmunoassay whereas tyrosine hydroxylase positive (TH+) neurons and PRL cells were morphometrically assessed in sections of fixed hypothalami and pituitaries, respectively. After 30 days, hypothalamic estrogen implants induced a significant increase in serum PRL, whereas TH+ neurons were not detectable in the arcuate-periventricular hypothalamic (ARC) region of estrogen-implanted rats. Removal of implants on day 30 restored TH expression in the ARC and brought serum PRL back to basal levels 30 days after estrogen withdrawal. Subcutaneous or i.m. administration of estrogen for 7 weeks induced a marked hyperprolactinemia. However, 30 weeks after estrogen withdrawal, TH neuron numbers in the ARC were back to normal and serum PRL returned to basal levels. After peripheral but not central estrogen withdrawal, pituitary weight and lactotrophic cell numbers remained slightly increased. Our data suggest that estrogen even at high doses, does not cause permanent damage to TIDA neurons.

The Expression of Estrogen, Prolactin, and Progesterone Receptors in Mammary Gland and Liver of Female Rats During Pregnancy and Early Postpartum: Regulation by Thyroid Hormones

The aim of this study was to examine, using semiquantitative reverse transcriptase–polymerase chain reaction (RT–PCR) the changes in mRNA expression of the two estrogen receptor (ER) subtypes, ERα and ERβ, prolactin receptor long and short form, and progesterone (Pg) receptor (PgR), in liver and mammary gland during gestation, early lactation, and weaning in both hyperthyroid (HT) and normal rats. Pregnancy increased long prolactin receptors (PRL-RL) and ERα mRNAs in liver and PRL-RL in mammary gland. Lactation decreased PRL-RL in liver and ERβ and PgR in mammary gland. HT decreased PRL-RL at the end of pregnancy (G21), ERα (in G21 and L1) in liver and PRL-RL in L1 as well as short prolactin receptors (PRL-RS) (G7, L1) and ERβ (G7, G14, L4) in mammary gland. In conclusion, our data indicated that PRL–R1 and ERα expression levels are differentially regulated in the liver, and PgR and ERβ in mammary gland during pregnancy and lactation ERβ is variably expressed depending on the state of thyroid hormones, however the ERα gene expression remained constant in mammary gland. PRL–R1 mRNA expression is highly induced in the mammary gland during late pregnancy and abruptly declines on the first day of lactation for the HT rats.

Hypothyroidism prolongs corpus luteum function in the pregnant rat

It has been shown that hypothyroidism in the rat produces a prolongation of pregnancy associated with a delay in the fall of circulating progesterone (P4) at term. The aim of the present work is to determine whether the delayed P4 decline in hypothyroid mother rats is due to a retarded induction of P4 degradation to 20alphaOH P4 or to a stimulation of its synthesis, and to investigate the possible mechanisms that may underlie the altered luteal function. We determined by RIA the circulating profile of the hormones (TSH, PRL, LH, P4, PGF2alpha, and PGE2) involved in luteal regulation at the end of pregnancy and, by semiquantitative RT-PCR, the expression of factors involved in P4 synthesis (CytP450scc, StAR, 3betaHSD, PRLR) and metabolism (20alphaHSD, PGF2alphaR, iNOS and COX2). Our results show that the delay in P4 decline and parturition is the resultant of retarded luteal regression, caused by a combination of decreases in luteolytic factors, mainly luteal PGF2alpha, iNOS mRNA expression and also circulating LH, and increased synthesis or action of luteotrophic factors, such as luteal and circulating PGE2 and circulating PRL. All these changes may be direct causes of the decreased 20alphaHSD mRNA and protein (measured by western blot analysis) expression, which in the presence of unchanged expression of the factors involved in P4 synthesis results in elevated luteal and circulating P4 that prolonged pregnancy and also may favor longer survival of the corpus luteum.

Effect of chronic thyroid hormone treatment on cycling, ovulation, serum reproductive hormones and ovarian LH and prolactin receptors in rats

We studied the effect on cycling, ovulation and hormone secretion of a chronic thyroxine treatment (HT, 1 mg/kg,S.C., daily, initiated at oestrus) on female rats. HT rats showed normal 4-day vaginal cycles on the first three cycles after initiation of the treatment, but on the fourth cycle had a prolonged oestrus and subsequently entered in constant di-oestrus. In spite of the normal vaginal cycles only 66%, 50%, 33% and 10% of the HT rats ovulated on cycles 1 to 4 respectively. In contrast, during cycles 2 and 3, ovulating HT rats shed a significantly greater number of ova than controls. Hormones were measured at 12.00 and 18.00 h (pre-ovulatory) on prooestrus and at 11.00 h on oestrus. HT ovulating rats had normal LH levels on the first two cycles, but low levels on the third one, while non-ovulating HT rats had low preovulatory LH levels. Serum FSH concentrations were elevated in all the HT rats on cycles 1 and 2 and on pro-oestrus morning in cycle 3 and may have been responsible for the increase in ovulation rate. On oestrus, ovulating HT rats had higher FSH values than nonovulating ones. Serum prolactin levels were similar to controls in all the HT rats on cycle 1, but on the subsequent cycles pre-ovulatory levels were lower than controls in all the HT rats, while values were increased in the non-ovulating HT rats on the third and fourth oestrus mornings. Pro-oestrous serum oestradiol concentrations in all the HT rats were not different from controls on cycles 1 and 2 and diminished on 3 and 4. Oestrous levels were significantly lower on the cycle 1 and only on the nonovulating HT rats on cycle 2. Serum progesterone levels had values similar to those of FSH, with increased values in the first two cycles. Serum corticosterone levels were increased in the mornings of cycles 2 and 3, but values were normal on the fourth one. Ovarian prolactin and LH receptor mRNAs, measured on HT rats on the third prooestrus by Northern blotting, showed significant increases in all the majoritary molecular forms (2.5 and 7 kb for LH receptor and 0.9, 2.9–3, 5 and 10 kb for the prolactin receptor) with respect to control pro-oestrous rats. These results show a progressive disruption of cycling, ovulation and hormonal secretion after the initiation of a chronic thyroid hormone treatment in rats, which eventually lead to an anovulatory state. These results may be of importance for the interpretation of the reproductive disfunctions provoked by hyperthyroidism in women.

Effect of chronic thyroxine treatment on pregnancy in rats: effects on oestrogen, progesterone, prolactin and GH receptors in uterus, liver and mammary gland.

We have previously shown that experimental hyperthyroidism produces premature and difficult delivery and absence of lactation in spite of apparently adequate luteolysis and lactogenesis. To study the possible causes of these alterations we measured the effect of treatment with T4 (0.25 or 1 mg kg(-1), s.c., daily, started 10-15 days before mating, HT0.25 and HT1) on serum hormones and their receptor (R) concentrations in reproductive tissues on day 20 of pregnancy (1800 hours), comparing them with controls on the same day (C20), or on day 21 of pregnancy (1800 hours) (C21). Serum prolactin (PRL) and corticosterone (B) concentrations increased in the HT groups, progesterone (Pg) and GH decreased and estradiol (E2) did not change, compared with C20 group. C21 rats had increased serum PRL and decreased Pg and GH. In HT rats mammary DNA and protein tissue content was doubled. Receptor concentrations were expressed per mg DNA. Mammary PRL-R were increased in HT1 rats, while E-R and Pg-R were significantly lower in both HT groups. HT0.25 and HT1 rats had increased uterine E-R and Pg-R and decreased liver PRL-R and GH-R as well as their mRNAs. Liver E-R, PRL-R and GH-R were decreased in C21 rats, while uterine Pg-R were increased. Thus, some of the observed changes (serum Pg and GH, mammary and uterine Pg-R, and liver GH-R and PRL-R decreases and serum PRL increase) may be due at least partially to the advancement in luteolysis and delivery, being similar to the changes observed between days 20 and 21. The changes in serum B, mammary PRL-R, and mammary and uterine E-R may be caused solely by the T4 treatments and may play a role in the alterations previously observed.

Seasonal reproduction in male pichis Zaedyus pichiy (Xenarthra: Dasypodidae) estimated by fecal androgen metabolites and testicular histology

Poaching poses a threat to a wide variety of wildlife, and basic information about the biology of hunted species needs to be collected before their populations decline to the extent that requires drastic human intervention. As the survival of a species is related to its ability to reproduce, data on its reproductive cycle are necessary for the development of management strategies. The hypothesis was tested that the reproductive season of pichis (Zaedyus pichiy), small hibernating armadillos that inhabit arid environments in Argentina and Chile, is limited to spring months. Gonadal competence of semi-captive and wild-caught male pichis of Mendoza Province, Argentina was studied, by measuring fecal immunoreactive testosterone concentrations and evaluating spermatogenic activity. Results suggest that Z. pichiy is a seasonal breeder that regulates reproduction through photoperiodic cues. Gonadal competence was limited to a period of 3-5 months in spring and early summer and was reflected in enlarged testes, increased spermatogenesis, and significantly elevated fecal immunoreactive testosterone concentrations. The reproductive season for males from southern Mendoza was almost 6 weeks shorter than in the north. This fact, along with significant morphological differences between both groups, suggests that northern and southern pichis belong to two distinct populations. It is concluded that prolonged breeding seasons and more favorable environmental conditions in northern Mendoza favor a prolongation of the reproductive season that may allow pichis to breed later in the year, thus maximizing reproductive opportunities.