UNIVERSIDADE FEDERAL DE SANTA MARIA CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM FARMACOLOGIA EFEITOS DO USO DO ACETATO DE LEUPROLIDE NAS CARATERÍSTICAS REPRODUTIVAS DE ROEDORES SUBMETIDOS AO TRATAMENTO COM TESTOSTERONA PROPIONATO PRÉ E PÓS-NATAL DISSERTAÇÃO DE MESTRADO Lady Katerine Serrano Mujica Santa Maria, RS, Brasil. 2016
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1
UNIVERSIDADE FEDERAL DE SANTA MARIA
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM FARMACOLOGIA
EFEITOS DO USO DO ACETATO DE LEUPROLIDE NAS CARATERÍSTICAS
REPRODUTIVAS DE ROEDORES SUBMETIDOS AO TRATAMENTO COM
TESTOSTERONA PROPIONATO PRÉ E PÓS-NATAL
DISSERTAÇÃO DE MESTRADO
Lady Katerine Serrano Mujica
Santa Maria, RS, Brasil.
2016
EFEITOS DO USO DO ACETATO DE LEUPROLIDE NAS CARATERÍSTICAS
REPRODUTIVAS DE ROEDORES SUBMETIDOS AO TRATAMENTO COM
TESTOSTERONA PROPIONATO PRÉ E PÓS-NATAL.
Por
Lady Katerine Serrano Mujica
Dissertação apresentada ao Curso de Mestrado do Programa de
Pós-Graduação em Farmacologia da Universidade Federal de Santa Maria
(UFSM, RS), como requisito parcial para obtenção do grau de.
Mestre em Farmacologia.
Orientador: Prof. Dr. Fabio Vasconcellos Comim
Coorientador: Prof. Dra. Melissa Orlandin Premaor
Santa Maria, RS, Brasil.
2016
3
UNIVERSIDADE FEDERAL DE SANTA MARIA
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO FARMACOLOGIA
A Comissão Examinadora, abaixo assinada,
Aprova a Dissertação de Mestrado
EFEITOS DO USO DO ACETATO DE LEUPROLIDE NAS CARATERÍSTICAS
REPRODUTIVAS DE ROEDORES SUBMETIDOS AO TRATAMENTO COM
TESTOSTERONA PROPIONATO PRÉ E PÓS-NATAL
Elaborada por
Lady Katerine Serrano Mujica
Como requisito parcial para obtenção do grau de
Mestre em Farmacologia
COMISSÃO EXAMINADORA:
______________________________________
Fabio Vasconcellos Comim PhD (UFSM)
(Presidente/Orientador)
______________________________________
Melissa Orlandin Premaor PhD (UFSM)
(Coorientador)
______________________________________
Edison Capp PhD (UFRGS)
______________________________________
Carlos Fernando de Mello PhD (UFSM)
Santa Maria, 30 de julho de 2016
5
AGRADECIMENTOS
Ao Criador por me acompanhar sempre.
A minha mãe Magdalena e meu pai Luís, os melhores pdo mundo, obrigada por
sempre caminharem de meu lado, por fazer de meus sonhos sempre realidade, ao meu
irmão de coração Raul pela cumplicidade, por sempre cuidar de mim da forma em que só
um irmão consegue fazer.
À minha família pelo apoio constante e incondicional, mesmo estando longe,
sempre senti sua presença a cada dia.
Aos Orientadores, Fabio Vasconcellos Comim e Melissa Orlandin por acreditar em mim,
pela confiança e sinceridade sempre.
Aos Professores, Paulo Bayard Dias Gonçalves, Alfredo Antoniazi e João Francisco
Coelho de Oliveira (in memoriam), pelo acolhimento na família BioRep.
Aos biorepianos da pós-graduação em especial aos colegas Vitor, Alessandra,
Mariana e Andressa pelo constante auxílio nos experimentos realizados; pela amizade que
será para sempre. Obrigada por cada um dos momentos vividos.
Aos biorepianos da graduação, com os quais pude contar com constante auxílio à
qualquer hora do dia.
À Karina Gutierrez e Werner Glanzner, obrigada pela amizade sincera, por cada
conselho e por serem as pessoas maravilhosas, pela luz que vocês têm e que sempre me
acompanhou nessa aventura.
Às minha família Brasileira, ao longo destes anos longe de minha família e de meus
pais, tive a oportunidade de conhecer pessoas maravilhosas que agora fazem parte da minha
vida, tios Domnina e Segefredo, nada seria possível sem vocês ao meu lado.
Aos amigos que deixei na Colômbia agradeço por que mesmo longe ficaram tão
perto, aos novos amigos que fiz de todas as partes do mundo, vocês fizeram meus dias mais
felizes. Muito obrigada por cruzar e estar sempre em meu caminho.
A CAPES, FAPERGS e ao CNPq pelo apoio financeiro para realização dos
experimentos.
É necessário abrir os olhos e perceber que as coisas boas estão dentro de nós, onde os
sentimentos não precisam de motivos nem os desejos de razão. O importante é aproveitar o
momento e aprender sua duração, pois a vida está nos olhos de quem saber ver.
Gabriel Garcia Márquez
7
RESUMO
Dissertação de Mestrado
Programa de Pós-Graduação em Farmacologia
Universidade Federal de Santa Maria
EFEITOS DO USO DO ACETATO DE LEUPROLIDE NAS CARATERÍSTICAS
REPRODUTIVAS DE ROEDORES SUBMETIDOS AO TRATAMENTO COM
TESTOSTERONA PROPIONATO PRÉ E PÓS-NATAL.
AUTOR: LADY KATERINE SERRANO MUJICA
ORIENTADOR: FABIO VASCONCELLOS COMIM
Data e Local da Defesa: Santa Maria 30 de julho de 2016.
A exposição a uma grande quantidade de androgênios pode induzir perturbações na função
reprodutiva e metabólica em ratas fêmeas que se assemelham a síndrome dos ovários policísticos
(SOP) em mulheres. Nestes animais, o grau de alteração na esteroidogênese e foliculogênese pode
variar de acordo com o período de intervenção, o que sugere diferentes mecanismos de atuação no
eixo hipotalâmico-pituitário, antes e após o nascimento. Neste estudo, utilizou-se um agonista de
GnRH, acetato de leuprolide, administrado por uma única injeção de depósito até 48h de vida em
ratas Wistar submetidos ao pré-natal e pós-natal protocolos androgenização à procura de seu
impacto reprodutivos. Androgenização pré-natal consistiu na administração de 2,5 mg de
testosterona propionato s.c. às mães nos dias 16, 17 e 18 (grupo pré-natal ou PreN), enquanto
androgenização pós-natal foi realizada através da injeção de 1,25 mg de testosterona propionato s.c
aos animais em 5 dias de vida (Grupo pós-natal ou PostN). Entre 90 e 110 dias de vida, estes
animais foram avaliados para os ciclos de estro (definida por citologia de série vaginal e a presença
do corpo lúteo), a produção de esteroides (níveis séricos de testosterona e androstenediona medida
por HPLC-MS / MS), morfologia dos ovários (histologia) e da expressão gênica hipotalâmica de
Kiss1, Gnrhr e GnRH. Observamos que no grupo PostN, caracterizado por anovulação e aumento
do número combinado de cistos e folículos atrésicos, que uma única injeção de acetato de leuprolide
depot (usualmente com duração de quatro semanas) foi capaz de aumentar no animal adulto as taxas
de ovulação estimadas por esfregaço vaginal. O tratamento com este agonista do GnRH também
reduziu os níveis de testosterona no soro e o número de cistos e folículos atréticos em PostN L em
contraste com os animais do grupo PostN (p = 0,04), ratos pré-natal androgenizadas (PreN)
exibiram alterações significativas nos genes hipotalâmicos, tais como uma redução de Kiss1 e
aumento da Gnrh, em comparação com grupos de controle e PostN. Em nosso conhecimento, este é
o primeiro estudo a mostrar que a administração de agonista de GnRH (acetato de leuprolide) em
dose única pode preservar com sucesso ciclos regulares, reduzir os níveis de androgênios e o
número de cistos / folículos atrésicos em ratas após o nascimento androgenizadas. Além disso, os
resultados obtidos neste modelo animal de SOP sugerem uma clara participação de anormalidades
no GnRH levando a anovulação e formação de cistos.
Palavras chave: síndrome do ovário policístico; programação de desenvolvimento; acetato de
leuprolide.
ABSTRACT
Master Course Dissertation
Professional Graduation Program in Pharmacology
Universidade Federal de Santa Maria
The Impact of Postnatal Leuprolide Acetate Treatment on Reproductive
Characteristics in a Rodent Model of Polycystic Ovary Syndrome
AUTHOR: LADY KATERINE SERRANO MUJICA
ADVISER: FABIO VASCONCELLOS COMIM
Defense Place and Date: Santa Maria, July 30nd
, 2016
Androgen excess induces in female rats reproductive and metabolic disturbances that are similar to
polycystic ovary syndrome in women. In these animals, the degree of changes may vary according
to the timeline intervention, suggesting that the hypothalamic and pituitary axis may present
different actions at pituitary-hypothalamic level before and after birth. The present study employed
an agonist GnRH, leuprolide acetate, administrated as a single dose upto 48h of life in Wistar rats
under prenatal or postnatal androgenisation protocols, looking for its effects on reproductive
endpoints. Prenatal androgenisation (PreN) consisted in the administration of testosterone
propionate 2.5 mg s.c. to mothers at the days 16,17 and 18, while postnatal androgenisation (PostN)
was performed through the injection of 1.25 mg s.c. to animals within 5 day of life. Between 90 -
100 days of life, these rats were evaluated to the present of estral cycles (defined by vaginal smears
and the presence of corpus luteum (CL), steroid levels (serum testosterone and androstenedione
measured by liquid chromatography (HLPC MS/MS), ovarian morphology (histology), and the
expression of mRNA for hypothalamic Kiss1, Gnrhr, and Gnrh and ovarian genes (Cyp17a1,
Cyp19a1, CYp11a1,and Gnrhr).We observed that in PostN group, characterized by anovulation and
increased number of cysts/atretic follicles), a single injection of a leuprolide acetate depot (lasting 4
weeks) (PostN L)was able to increase ovulatory rates, as defined by vaginal smears. The treatment
with this GnRH agonist in PostN L also reduced testosterone serum levels as well the number of
cysts/atretic follicles in contrast with PostN group (p=0.04). Prenatally androgenized rats (PreN)
exhibited significant abnormalities at hypothalamic genes, such as a reduction of Kiss1 and
increased of Gnrh,in comparison with control and PostM groups. To the best of our knowledge,
this is the first study to show that previous administration of a single dose of GnRH agonist
(leuprolide acetate) could successfully preserve regular cycles, reduce androgen levels and the
number of cysts/atretic follicles in rats submitted to androgen excess postnatally. Indeed, the results
obtained with this model of PCOS suggest a clear participation of GnRH disruption to the progress
Polycystic ovary syndrome (PCOS) is one of the most frequent causes of anovulatory 51
infertility, affecting 5%–10% of women of reproductive age worldwide. PCOS is an 52
endocrine disorder characterized by hyperandrogenism and ovarian abnormalities related to a 53
disruption in the hypothalamic–pituitary–ovarian axis (Baptiste et al. 2010; Davies et al. 54
2011; Franks 2012; Glintborg 2016). Growing evidence in the literature has suggested that 55
changes occurring during gestation or even early after birth could be related to many 56
reproductive conditions that replicate PCOS features in adult life (Barker 1990; Franks 2012). 57
Indeed, rhesus monkeys, sheep, and rodents submitted to exposure to high levels of androgens 58
or aromatase inhibitors demonstrate biochemical hyperandrogenism or chronic anovulation in 59
adult life (Abbott et al. 2005; Birch et al. 2003; Chinnathambi et al. 2012; Franks 2012; 60
Manneras et al. 2007; Ortega et al. 2009; Padmanabhan and Veiga-Lopez 2013; Walters et al. 61
2012; West et al. 2001). In rats, distinct phenotypes are observed after prenatal or postnatal 62
androgenization protocols. In most of the studies, the exposure to androgens during the 63
prenatal period is associated with an increased level of testosterone in adulthood 64
(Chinnathambi et al. 2012; Daneshian et al. 2015; Tehrani et al. 2014; Wu et al. 2010). 65
Nevertheless, not frequently identified are other features suggestive of PCOS: rats submitted 66
to fetal programming (prenatal protocols) usually demonstrated the persistence of estrus 67
cycles (Tehrani et al. 2014; Tyndall et al. 2012; Wu et al. 2010) and almost invariably showed 68
normal ovary size (Tyndall et al. 2012; Wu et al. 2010). As a rule, the appearance of ovaries 69
from rats treated with androgens prenatally is not significantly different than that of controls; 70
just a few studies have reported the multicystic appearance of ovaries (Fels and Bosch 1971; 71
Slob, et al. 1983; Tyndall et al. 2012; Wu et al. 2010). In contrast to fetal programming, 72
postnatal rodent models of PCOS are usually characterized by disrupted ovulation (Anderson, 73
et al. 1992; Huffman and Hendricks 1981; Lee, et al. 1991; Tyndall et al. 2012; Weisz and 74
Lloyd 1965), development of cysts, decrease or absence of corpus luteum ovulation 75
(Anderson et al. 1992; Huffman and Hendricks 1981; Lee et al. 1991; Tyndall et al. 2012; 76
Weisz and Lloyd 1965), and increase of androgen levels (Lee et al. 1991; Marcondes, et al. 77
2015; Weisz and Lloyd 1965). In this hypothesis-generating study, we investigated the effects 78
of GnRH agonist (acetate leuprorelin) in rats submitted to an androgen excess before and after 79
birth. As shown below, the treatment before 48h of life with a single injection of Leuprolide 80
acetate depot (lasting 3-4 weeks) was able to increase ovulatory rates and reduce the cysts and 81
atretic follicles in postnatal androgenized rat model. Even so, it did not change androgen 82
levels or hypothalamic (Kiss1, GnRH) or ovarian (CYP17a1, CYP11a1, and STAR) 83
expression of key genes. These results contrasted with the prenatal model of treatment with 84
leuprolide (PreN L), which exhibited a small improvement in estrus cycles (not affecting the 85
corpus luteum or atretic follicle number), associated with a significant modification of the 86
expression of Kiss1 and GnRH genes in the hypothalamus. 87
MATERIALS AND METHODS 88
Animals 89
This study was approved by the Ethics Committee on Animal Use (CEUA) the Federal 90
University of Santa Maria (UFSM), Brazil, under protocol number 100/14. Overall, thirty 91
females and twenty males Wistar rats (Rattus norvegicus albinus) aged between 70 days were 92
used in this study and were housed at the Laboratory Animal Reproduction (BioRep) the 93
Federal University of Santa Maria (UFSM). The animals were maintained at a temperature of 94
220C, 55 – 65% humidity under artificial illumination on a light–dark cycle of 12:12 h, with 95
daylight from 7 a.m. to 7 p.m. Food and water were given ad libitum. 96
Synchronization of estrus and Treatment Protocols 97
39
Thirty female rats were submitted to the protocol for synchronization of estrus. They received 98
an i.p. injection of 10 IU of equine chorionic gonadotropin (eCG; Folligon™, Intervet, 99
Brazil), followed 48 h later by 10 IU of human chorionic gonadotropin (hCG; Pregnyl™, 100
Organon, Brazil), and were placed with a male for 24 hours (Agca, et al. 2013). Female rat 101
pups were divided into four groups for treatment of testosterone propionate androgenization 102
and three control groups. Dams were maintained with their pups until weaning (21 days). 103
Prenatal hormone exposure was accomplished by treatment of pregnant dams from 104
Embryonic Days 16, 17, and 18 injection s.c. of 2.5mg testosterone propionate 105
(Androgenol™, Hertape Calier, Brazil) (PreN group), while vehicle control exposures were 106
accomplished by treatment of pregnant dams from Embryonic Days 16, 17, and 18 injection 107
s.c. 2.5 mg corn oil (Control PreN). Postnatal hormone exposures were performed by the 108
treatment of 5-day-old animals through a subcutaneous injection of 1.25 mg testosterone 109
propionate (PostN group), while vehicle control postnatal 5-day-old animals received a 110
subcutaneous injection of 1.25 mg corn oil (Control PostN). The treatment with leuprolide 111
acetate was realized in 2-day-old animals through an intermuscular injection of 0.40 mg 112
leuprolide acetate depot (Lectrum™, Sandoz, Brazil) in prenatal androgenized (PreN L) and 113
postnatal androgenized (PostN L) (Fig. 1). A dose of leuprolide was based on its use for 114
infants with precocious puberty, i.e., 0.3 mg/kg (usually doses of attack may be higher). A 115
vehicle control was given to 2-day-old animals through an intermuscular injection of 0.40 mg 116
corn oil (Control L). Prenatal androgenization was obtained through a previous protocol, 117
including the administration of 2.5 mg of testosterone propionate on days 16–18 of gestation 118
(Wu et al. 2010), while postnatal androgenization was treated with 1.25 mg of testosterone 119
propionate s.c. 5-day-old female pups (Swanson and Werfftenbosch 1964). Groups at the end 120
of the study were prenatal (PreN n = 8), postnatal (PostN n = 7), control prenatal 121
(ControlPreN n =4), control postnatal (ControlPostN =4), prenatal-leuprolide (PreN L n = 4), 122
postnatal-leuprolide (PostN L n = 8), and control leuprolide (Control L n = 8). For analysis of 123
gene expression of the ovary and hypothalamus, all control groups were included as a one. 124
Estrus cycle 125
Vaginal smears were collected on glass slides to evaluate the animal’s estrus cycles from 90 126
days to 100 days of age. Panótico™ (Laborclin, Brazil) staining was used to analyze the 127
vaginal cytology. Cytology was examined by a "blind" examiner (K.B.) with experience in 128
this procedure. A normal estrus cycle was defined as exhibiting all phases (proestrus, estrus, 129
metestrus, and diestrus) over a period of 4–5 days, as previously characterized in the 130
literature. In proestrus, oval nucleated epithelial cells, occasionally with a small number of 131
keratinocytes, were detected. In estrus, epithelial keratinocytes with irregular shapes were 132
detected; they resembled deciduous leaves or were interconnected into pieces, among which 133
there was a small number of nuclear epithelial cells. In metestrus, irregular epithelial 134
keratinocytes, nucleated epithelial cells, and leukocytes were detected. In diestrus, a large 135
number of leukocytes and a small number of nuclear epithelial cells were detected. 136
Euthanasia 137
At 110 days of age, the animals were transferred and then anesthetized with isoflurane plus 138
administration of tramadol chloride (Tramadol™, Pfizer, Brazil) i.m. (20-40 mg/kg). The 139
minimum alveolar concentration (MAC) of isoflurane used for anesthesia of the rats was 140
estimated at 5%, at a laboratory temperature established artificially between 20 and 25C (or 141
about 31.7 kPa or 238 mm Hg to 39.3 kPa 295 mm Hg). Between 9:00 a.m. and 13:00 p.m., 142
blood samples were collected, and the ovaries were removed before the animals were finally 143
euthanized with cardiac puncture under deep anesthesia in the absence of pedal and corneal 144
reflexes. 145
Hormonal dosage through UHPLC-MS/MS 146
The identification and quantification of androstenedione and testosterone were also performed 147
by a blind examiner using Ultra High-Performance Liquid Chromatography Tandem Mass 148
Spectrometry (UHPLC-MS/MS), as detailed below. The UHPLC-MS/MS was from Waters 149
(USA), equipped with Acquity UPLC™ liquid chromatography; a Xevo TQ™ MS/MS triple 150
quadrupole detector; an autosampler, a binary pump, and a column temperature controller; 151
and a nitrogen generator, model NM30L-MS (Peak Scientific, Scotland), with argon gas 6.0 152
used as collision gas. Data acquisition was realized employing the software MassLynx V4.1. 153
For chromatographic separation, an analytical column Acquity UPLC™ BEH C18 (100×2.1 154
mm, 1.7-μm particle size), maintained at 40°C, was used. For extract preparation, 200µL 155
samples were diluted in acetonitrile until reaching 800µL. This mixture was homogenized by 156
30 seconds vortexing and injected in the UHPLCMS/MS system. After that, an analytical 157
solution (internal pattern) for androstenedione and testosterone was added to the mixture to 158
reach a final concentration of 50ng L-1. This new mixture was once again injected into the 159
system. The linearity and the detection limits of each analytic were verified through analytic 160
solutions with concentrations ranging from 50 to 5000ng L-1 injected in the UHPLC-MS/MS. 161
41
Using the data obtained with dilutions, the calibration curves were obtained. The 162
spectrophotometer was operated in the selected reaction monitoring (SRM) mode, with two 163
transitions for each analytic, one for quantification and the second for confirmation. The 164
higher intensity transition corresponds to analytic quantification and the second higher 165
transition corresponds to confirmation. The ionization mode used was Electrospray Ionization 166
(ESI) in positive mode for androstenedione and testosterone, with column oven temperature at 167
40°C, the pressure of 15000psi, capillary of 2.8kV, desolvation temperature at 500°C, gas 168
flow 800L h-1, collision gas flow (argon) of 0.15mL min-1 and source temperature about 169
150°C. The mobile phase used was composed of aqueous solution (solvent A), 0.05% of 170
ammonium hydroxide and methanol (solvent B). A boiling gradient was used with 0.150mL 171
min-1 flow and an injection volume of 10µL and total running time of 5min (Rigo, et al., 172
2015). 173
Morphological and morphometric analyses of the ovaries 174
The ovaries were fixed in 4% paraformaldehyde for 24h. They were then submitted to the 175
following steps: ethyl alcohol dehydration, diaphanization in xylene, liquid Paraplast™ 176
(Sigma, Brazil), impregnation in a drying stove at 60°C, and Paraplast™ inclusion at room 177
temperature. The Paraplast blocks containing the tissues were sectioned using a microtome 178
(RM2335, Leica Biosystems), adjusted for sections of 5 µm thickness on glass slides. 179
Afterward, each slide was stained with hematoxylin and eosin, as previously published 180
(Comim F et al. 2015). The morphological and morphometric analyses (follicle and corpora 181
lutea counting) were realized independently by two blind examiners (F.V.C and F.C.D.), 182
using as tools for identification (Dixon, et al. 2014; Kang, et al. 2015) an optical microscope 183
(Leica, DMI4000B) coupled with a digital camera system. In the case of discordance, a third 184
research (LEM) was called to define the final result. Overall, the Cohen´s kappa index 185
(agreement between the examiners) was superior to 0.9. 186
RNA extraction and cDNA synthesis 187
Ovarian and hypothalamic total RNA extraction was performed using organic extraction 188
methods Trizol® (Life Technologies, Foster City, CA, United States), following the 189
manufacturer’s instructions. The samples were treated with DNase I™ (Life Technologies, 190
United States). The RNA samples were quantified in a spectrophotometer NanoDrop (ND 191
1000, Thermo Scientific) with a wavelength of 260nm. Total RNA (1μg) was treated with 192
DNase™ (DNAse Amplification Grade I - Invitrogen) at 37oC within 5 minutes to digest any 193
contaminating DNA. The reverse transcription reaction to cDNA was performed using the 194
iScript cDNA Synthesis Kit™ (Bio-Rad) according to the manufacturer's instructions. cDNAs 195
were kept in a –20°C freezer until the gene expression was checked. Its efficiency was 196
verified via a quantitative real-time polymerase chain reaction (qRT-PCR) when the gene 197
expression was evaluated. β-actin (NM_031144.2) and cyclophilin-A (NM_017101.1) 198
specific for Rattus norvegicus were used as possible endogenous controls. The genes of 199
interest were CYP11a1 (NM_007264.2), CYP17a1 (NM_012753.1), CYP19a1 200
(NM_007810.3), and Gnrhr (NM_031038.3) in ovarian tissue and Gnrh (NM_012767.2), 201
Gnrhr (NM_031038.3), and Kiss1 (NM_181692.1) in hypothalamic tissue. Details of primers 202
used in this study are disposed in (Fig. S1). The qRT-PCR reactions were performed in 203
duplicate in a total volume of 20 µL (including 10 ng of cDNA, 10 µL of TaqMan Universal 204
PCR Master Mix, and 1 lµ of the assays) and were carried out on CFX384 real-time PCR 205
systems (Bio-Rap) (Hercules, CA). The cycle conditions were as follows: 50°C for 2 min, 206
95°C for 10 min, 45 cycles of 95°C for 10 s, and 60°C for 1 min. The data were analyzed 207
using Bio-rap CFX Manager Software (version 3.0), and Ct values were transformed to 208
quantities using the comparative Ct method (DDCt, Life Technologies). 209
Statistical analysis 210
The statistical analysis was performed using the software GraphPad Prism 6.03 (GraphPad 211
Software Inc., San Diego, CA). Comparisons among the groups were performed, using 212
ANOVA and, then, followed by post hoc comparisons, using the Bonferroni test. In the 213
absence of a normal distribution, the data were analyzed, using a Kruskal–Wallis test, 214
followed by Dunn’s post hoc test. Proportion among groups was compared, using the Fisher´s 215
test. Significance was assumed at P<0.05. 216
RESULTS 217
Estrus cycle 218
Control, Control L, PreN, and PostN L began to present estrus cycles on the 60th day, which 219
became regular only on the 70th day. In the prenatal group (PreN), exposure to testosterone 220
in the uterus led to a partial closure of the vagina; however, it still allowed the collection of 221
material for cytology analysis. A proportion of the animals' estrus cycles improved (from 12 222
to 25%) after treatment with leuprolide acetate (Fig. 2). As expected, all control groups had 223
43
normal estrus cycles (4–5 days). The postnatally androgenized group (PostN) did not exhibit 224
any estrus cycle and remained at the metestrus and diestrus phases most of the time (Fig. S1- 225
S6). Nevertheless, the treatment of leuprolide acetate in the post-natal group preserved a 226
regular cycle capacity as shown in (Fig. 2). 227
Ovarian morphology 228
The rat ovaries were analyzed for the presence of corpus luteum, healthy antral follicles, and 229
cysts/atretic follicles. As shown in (Fig. 3D), a significant reduction in the number of corpus 230
luteum (per animal) was identified in the Post N group in comparison to the PreN and Control 231
groups. In the group treated with leuprolide (PostN L), the presence of corpus luteum was 232
identified, but it did not reach significance (p=0.056) (Fig. 3B). No differences in the number 233
of corpus luteum were noted between PreN rats in comparison to controls, or PreN L (Fig. 3D 234
and 3H). Cysts were significantly more detected in PostN animals than PreN or controls (Fig 235
3C- Remarkably, the treatment with leuprolide acetate reduced the number of cysts/atretic 236
follicles in the PostN L group (Fig. 3F). A trend to reduce healthy antral follicles (p=0.054) 237
was identified in controls treated with leuprolide acetate (Fig. 3J), although it did not affect 238
the number of corpus luteum or cysts/atretic follicles. 239
Androgen levels (testosterone and androstenedione) 240
Fig. 4. illustrated the hormonal profiles of female of experimental and control groups. The 241
serum testosterone and androstenedione levels in the groups PreN, PostN, and Control were 242
similar. Of note, testosterone levels significantly decreased only in the PostN L group 243
(p=0.04) (Fig. 4B). 244
Gene expression in ovary and hypothalamus 245
Ovary 246
CYP17a1 expression was higher in the PreN group against PostN or Control (p=0.01) (Fig 247
5H). No significant changes in CYP11a1 or CYP19a1 were noted regarding the type of 248
androgenization or treatment or not with leuprolide acetate (Fig. 5A-D and I-L). The 249
expression of Gnrhr was significantly higher in PreN than in the Control or PostN groups 250
(Fig. 5P). In the group treated (PreN L), an even greater increment of Gnrhr was observed 251
(Fig. 5N). 252
253
Hypothalamus 254
In the hypothalamus, Gnrh gene expression was significantly higher in the PreN group than in 255
the PostN or Control groups (p=0.01) (Fig. 6l). The expression of its receptor (Gnrhr) was 256
increased in PostN in comparison to PreN or Control (p=0.01) (Fig. 6H). Conversely, Kiss1 257
was decreased in PreN against Control (Fig. 6D), although an increase in expression of Kiss1 258
was demonstrated in the PreN L group (p=0.038) (Fig. 6B). The treatment with leuprolide 259
modified Gnrh in controls, increasing its gene expression (p=0.03) (Fig. 6I). No changes were 260
observed in PostN at any moment (Fig. 6C, G, and K). 261
DISCUSSION 262
The present study compared two protocols for androgenisation (pre and postnatal) in the 263
female Wistar rats looking for their reproductive impact at adult life. Also, it is evaluated 264
whether treatment with a GnRH agonist (Leuprolide acetate) at neonatal period could affect 265
the expected phenotype in these animal models of PCOS. Our results which replicated 266
previous findings in the literature (Walters et al. 2012), confirmed that androgen excess 267
exposition at a particular timeline might lead to different reproductive phenotypes. PreN 268
animals, submitted to androgen excess during gestation, exhibited ovulatory cycles, 269
comparable ovarian morphology to controls, but some degree of hyperandrogenism, as 270
defined by higher expression of cyp17a1 mRNA (androgen levels were not elevated). These 271
PreN group rats developed marked disruption of hypothalamic kiss1 and GnRH. However, 272
animals that were submitted to post-natal androgenisation (PostN) presented 100% of 273
anovulatory cycles and significant changes in ovarian morphology (decrease of corpus luteum 274
e increase of cysts/atretic follicles) with no evidence of hyperandrogenism. Data from this 275
study may suggest a role of GnRH in the development of ovarian and hypothalamic 276
abnormalities in PCOS, given the fact that blocking GnRH receptors through leuprolide 277
acetate during four weeks modified the development of anovulation and cysts and reduced 278
previous testosterone levels in PostN L group. In the elegant work of Tyndall et al. (2012), 279
different schemes of treatments with testosterone propionate (TP) were performed in Wistar 280
rat. Interestingly, the group late postnatal (TP from d15 to 24 of life), group fetal plus 281
postnatal TP (from fetal e15 to e21 and then d1 to 24 of life) and fetal (TP from d14.5 to 21.5) 282
were not capable to produce similar changes to those observed with full postnatal group (TP 283
from d1 to 24). These rats, like our PostN group, developed anovulation and lack of corpus 284
luteum and no evidence hyperandrogenism. Why earlier and full postnatal treatments with TP, 285
45
but not late postnatal treatments with TP could induce changes in the morphology of the ovary 286
and ovulatory rates, suggest the possibility of conditional timeline for reproductive changes. It 287
has been described in rats, during the 30 initial days of life, a physiological condition of 288
higher activation of GnRH axis, that would correspond to the mini puberty in humans. 289
Currently, the meaning of mini puberty in the development of PCOS is uncertain. The mini 290
puberty in boys is usually shorter (0-6 months) and shows LH levels higher than those of 291
girls, who have longer periods of activation of GnRH (up to two years) due to fluctuations of 292
estradiol and predominant FSH over LH (Anderson, et al. 1998; Kuiri-Hanninen et al. 2014; 293
Kuiri-Hanninen, et al. 2011). A study in sheep performed by Recabarreni et al. described that, 294
between 5-10 weeks, the pituitary secretion of LH in response to a short test with leuprolide 295
was higher (and not progressive) in prenatally androgenized ewes in comparison to controls. 296
At 20 and 30 weeks of age, the controls exhibited a significantly higher response to leuprolide 297
(Recabarren, et al. 2005). In female Sprague-Dawley rats, the responsivity of GnRH agonists 298
started to rise on day five of life, reached a peak on day 20, and decreased after the 30th day 299
(Wilkinson and Moger 1981). Another relevant point is that folliculogenesis in rodents, 300
differently from other mammals such ewes and human, is not complete before birth and 301
continues for few days after birth. Therefore, is more plausible that treating rats just after birth 302
may disrupt ovary development in progress. In our study, indirect evidence for 303
hyperandrogenism, a key manifestation of PCOS was manifest only in the PreN group. These 304
prenatally androgenized animals shown an affected gonadal axis identified by changes of 305
hypothalamic (GnRH, Kiss1, and Gnrhr) and ovarian genes (CYP17a1 and Gnrhr). Literature 306
has pointed out that prenatal T excess may reprogram hypothalamic secretion of GnRH and 307
its neuronal networking (Padmanabhan and Veiga-Lopez 2011). Prenatal androgen excess in 308
mice usually follows higher levels of LH associated with high GABAergic transmission 309
(Roland, et al. 2010; Sullivan and Moenter 2005); in ewes, prenatal androgenization leads to 310
an LH hypersecretion and modification of negative steroid feedback culminating with 311
anovulation at the second breed season (Cheng, et al. 2010; Robinson, et al. 1999; Unsworth, 312
et al. 2005; Veiga-Lopez, et al. 2009). Studies on sheep show that in KNDy neurons, the 313
expression of neurokinin B and the inhibitory dynorphin decrease in the ARC neurons while 314
Kiss remain in the same proportion of cells, leading to a higher secretion of Gnrh. In rats, 315
where a sexual dimorphism is present in KNDy neurons, the GnRH pulse frequency 316
determines LH but not FSH (McNeilly et al. 2003). Prenatal treatment (most of the time with 317
DHT or T at higher levels) leads to increased levels of LH associated with disrupted ovulation 318
(Walters et al. 2012). Nevertheless, why PreN rats exhibited great modifications in 319
hypothalamus without great disruption of estrus cycles should be clarified by further studies. 320
Our results show that treatment with leuprolide acetate had also led to changes in the control 321
group but that modifications were similar to those reported in mice. Similar to what is 322
described in this study, Singh and Krishna (2010) have shown that the use of a moderate dose 323
of GnRH agonist in healthy mice could lead to a reduction in late (healthy) antral follicles. 324
Another secondary hypothesis addressed in this study relies on the description of the disrupted 325
gene expression of Gnrhr in the ovary of prenatally androgenized rats. In the rat and mouse 326
ovary, Gnrhr mRNA are present in the granulosa, cumulus cells, and oocytes and suffer 327
effects from local GnRH production, exhibiting fluctuation during the estrus cycle (higher in 328
diestrus and proestrus phases) (Schirman-Hildesheim, et al. 2005; Torrealday, et al. 2013). 329
Whether treatment with leuprolide interfered with GnRHR and its actions on steroidogenesis 330
and folliculogenesis remains to be elucidated. We identify some limitations in our study. 331
Firstly, the phase of the estrus cycle was not addressed before euthanasia. It is known that the 332
expression of GnRH and its receptors (GnRHR) in the reproductive tissues of rats may vary 333
through the stages of the estrus cycle. GnRH usually peaks in the hypothalamus in proestrus, 334
while GnRHR is progressively higher during diestrus and suffers two consecutive peaks in 335
proestrus, reducing its expression in estrus. However, the histologic analysis demonstrated a 336
very similar and superposed ovulatory profile between controls and PreN rats, in respect of 337
the significant differences in the hypothalamic and ovary genes evaluated. Secondly, the 338
possible changes in the steroid receptor balance were not explored. It was not clear whether or 339
not the actions of androgens in the negative feedbacks of estradiol and progesterone were 340
affected by the treatment that used the GnRH agonist. Treating female mice with the 341
leuprolide acetate also modified the neurosteroid production of progesterone (Micevych et al., 342
2003). Thirdly, it was not possible to understand whether the effects of the leuprolide acetate 343
occurred directly at the hypothalamus or ovary or indirectly through the reduction of estradiol 344
levels. Similar doubts had been described in other studies that employed metformin in mice 345
(Roland et al., 2010) or rats (Liu et al., 2015). In spontaneously ovulating rodents, the 346
preovulatory LH surge is initiated on the day of proestrus; present studies explore whether or 347
not kisspeptin is part of the essential neural circuit that links to the GnRH system to stimulate 348
ovulation. In the gonadotropin-releasing hormone (GnRH), neurons stimulate the release of 349
peptide and activate the reproductive axis in mammals. Kisspeptin has pronounced pre- and 350
post-synaptic effects, with the latter dominating the excitability of the GnRH neurons (Clarke 351
et al., 2015; Ronnekleiv & Kelly, 2013), further suggesting that the direct activation of the 352
47
neurons is responsible for the release of the luteinizing hormone (LH) and the follicle-353
stimulating hormone (FSH) (IWASA et al., 2016). 354
To summarize, the present study adds, for the first time, new evidence of the actions of a 355
GnRH agonist depot in the androgenized model of rat pups (model of PCOS). Treatment with 356
a GnRH agonist up to 48h after birth demonstrates improvement of the estrus cycles, 357
reduction in cystic/atretic follicles, and decreased serum testosterone in the postnatally 358
androgenized rat. The impact in the prenatally androgenized rat was present but slight, 359
regarding ovulation. Therefore, we speculated that this period of life described as minipuberty 360
in humans and corresponding to the first month of life in rats could also be a suitable time for 361
target reproductive disruption. Further studies are necessary to elucidate the mechanisms 362
operating these changes, in particular regarding the negative feedback to steroids at the 363
hypothalamus under treatment with GnRH agonists. 364
365
REFERENCES 366
Abbott DH, Barnett DK, Bruns CM & Dumesic DA 2005 Androgen excess fetal programming of female 367 reproduction: a developmental aetiology for polycystic ovary syndrome? Hum Reprod Update 11 368 357-374. 369 370 Adewale HB, Jefferson WN, Newbold RR & Patisaul HB 2009 Neonatal bisphenol-a exposure alters rat 371 reproductive development and ovarian morphology without impairing activation of gonadotropin-372 releasing hormone neurons. Biol Reprod 81 690-699. 373 374 Agca C, Yakan A & Agca Y 2013 Estrus synchronization and ovarian hyper-stimulation treatments 375 have negligible effects on cumulus oocyte complex gene expression whereas induction of ovulation 376 causes major expression changes. Mol Reprod Dev 80 102-117. 377 378 Anderson E, Lee MT & Lee GY 1992 Cystogenesis of the ovarian antral follicle of the rat: 379 ultrastructural changes and hormonal profile following the administration of 380 dehydroepiandrosterone. Anat Rec 234 359-382. 381 382 Andersson AM, Toppari J, Haavisto AM, Petersen JH, Simell T, Simell O & Skakkebaek NE 1998 383 Longitudinal reproductive hormone profiles in infants: peak of inhibin B levels in infant boys exceeds 384 levels in adult men. J Clin Endocrinol Metab 83 675-681. 385 386 Baptiste CG, Battista MC, Trottier A & Baillargeon JP 2010 Insulin and hyperandrogenism in women 387 with polycystic ovary syndrome. J Steroid Biochem Mol Biol 122 42-52. 388 389 Barker DJ 1990 The fetal and infant origins of adult disease. BMJ 301 1111. Barraclough CA 1961 390 Production of anovulatory, sterile rats by single injections of testosterone propionate. Endocrinology 391 68 62-67. 392
Birch RA, Padmanabhan V, Foster DL, Unsworth WP & Robinson JE 2003 Prenatal programming of 393 reproductive neuroendocrine function: fetal androgen exposure produces progressive disruption of 394 reproductive cycles in sheep. Endocrinology 144 1426-1434. 395 396 Clarke H, Dhillo WS & Jayasena CN 2015 Comprehensive Review on Kisspeptin and Its Role in 397 Reproductive Disorders. Endocrinol Metab (Seoul) 30 124-141. 398 399 Comim FV, Hardy K, Robinson J & Franks S 2015 Disorders of follicle development and 400 steroidogenesis in ovaries of androgenised foetal sheep. J Endocrinol 225 39-46. 401 402 Cheng G, Coolen LM, Padmanabhan V, Goodman RL & Lehman MN 2010 The kisspeptin/neurokinin 403 B/dynorphin (KNDy) cell population of the arcuate nucleus: sex differences and effects of prenatal 404 testosterone in sheep. Endocrinology 151 301-311. 405 406 Chinnathambi V, Balakrishnan M, Yallampalli C & Sathishkumar K 2012 Prenatal testosterone 407 exposure leads to hypertension that is gonadal hormone-dependent in adult rat male and female 408 offspring. Biol Reprod 86 137, 131-137. 409 410 Daneshian Z, Ramezani Tehrani F, Zarkesh M, Norooz Zadeh M, Mahdian R & Zadeh Vakili A 2015 411 Antimullerian hormone and its receptor gene expression in prenatally androgenized female rats. Int J 412 Endocrinol Metab 13 e19511. 413 414 Davies MJ, Marino JL, Willson KJ, March WA & Moore VM 2011 Intergenerational associations of 415 chronic disease and polycystic ovary syndrome. PLoS One 6 e25947. 416 417 Dixon D, Alison R, Bach U, Colman K, Foley GL, Harleman JH, Haworth R, Herbert R, Heuser A, Long G, 418 et al. 2014 Nonproliferative and proliferative lesions of the rat and mouse female reproductive 419 system. J Toxicol Pathol 27 1S-107S. 420 421 Fels E & Bosch LR 1971 Effect of prenatal administration of testosterone on ovarian function in rats. 422 Am J Obstet Gynecol 111 964-969. 423 424 Foecking EM, McDevitt MA, Acosta-Martinez M, Horton TH & Levine JE 2008 Neuroendocrine 425 consequences of androgen excess in female rodents. Horm Behav 53 673-692. 426 Franks S 1995 Polycystic ovary syndrome. N Engl J Med 333 853-861. 427 428 Franks S 2006 Candidate genes in women with polycystic ovary syndrome. Fertil Steril 86 Suppl 1 S15. 429 Franks S 2012 Animal models and the developmental origins of polycystic ovary syndrome: increasing 430 evidence for the role of androgens in programming reproductive and metabolic dysfunction. 431 Endocrinology 153 2536-2538. 432 433 Glintborg D 2016 Endocrine and metabolic characteristics in polycystic ovary syndrome. Dan Med J 434 63. 435 Huffman L & Hendricks SE 1981 Prenatally injected testosterone propionate and sexual behavior of 436 female rats. Physiol Behav 26 773-778. 437 438 Iwasa T, Matsuzaki T, Tungalagsuvd A, Munkhzaya M, Yiliyasi M, Kato T, Kuwahara A & Irahara M 439 2016 Effects of chronic testosterone administration on body weight and food intake differ among 440 pre-pubertal, gonadal-intact, and ovariectomized female rats. Behav Brain Res 309 35-43. 441
Kafali H, Iriadam M, Ozardali I & Demir N 2004 Letrozole-induced polycystic ovaries in the rat: a new 442 model for cystic ovarian disease. Arch Med Res 35 103-108. 443
49
Kang X, Jia L & Shen X 2015 Manifestation of Hyperandrogenism in the Continuous Light Exposure-444 Induced PCOS Rat Model. Biomed Res Int 2015 943694. 445
Kuiri-Hanninen T, Sankilampi U & Dunkel L 2014 Activation of the hypothalamic-pituitary-gonadal 446 axis in infancy: minipuberty. Horm Res Paediatr 82 73-80. 447 448 Kuiri-Hanninen T, Seuri R, Tyrvainen E, Turpeinen U, Hamalainen E, Stenman UH, Dunkel L & 449 Sankilampi U 2011 Increased activity of the hypothalamic-pituitary-testicular axis in infancy results in 450 increased androgen action in premature boys. J Clin Endocrinol Metab 96 98-105. 451 452 Lee MT, Adams WC & Bruot BC 1991 Circulating hormone concentrations in hypothyroid rats with 453 induced polycystic ovaries. Proc Soc Exp Biol Med 198 737-741. 454 455 Liu W, Liu W, Fu Y, Wang Y & Zhang Y 2015 Bak Foong pills combined with metformin in the 456 treatment of a polycystic ovarian syndrome rat model. Oncol Lett 10 1819-1825. 457 458 Manneras L, Cajander S, Holmang A, Seleskovic Z, Lystig T, Lonn M & Stener-Victorin E 2007 A new 459 rat model exhibiting both ovarian and metabolic characteristics of polycystic ovary syndrome. 460 Endocrinology 148 3781-3791. 461 462 Marcondes RR, Carvalho KC, Duarte DC, Garcia N, Amaral VC, Simoes MJ, Lo Turco EG, Soares JM, Jr., 463 Baracat EC & Maciel GA 2015 Differences in neonatal exposure to estradiol or testosterone on 464 ovarian function and hormonal levels. Gen Comp Endocrinol 212 28-33. 465 466 McNeilly AS, Crawford JL, Taragnat C, Nicol L & McNeilly JR 2003 The differential secretion of FSH and 467 LH: regulation through genes, feedback and packaging. Reprod Suppl 61 463-476. 468 469 Micevych PE, Rissman EF, Gustafsson JA & Sinchak K 2003 Estrogen receptor-alpha is required for 470 estrogen-induced mu-opioid receptor internalization. J Neurosci Res 71 802-810. 471 472 Ortega HH, Salvetti NR & Padmanabhan V 2009 Developmental programming: prenatal androgen 473 excess disrupts ovarian steroid receptor balance. Reproduction 137 865-877. 474 475 Padmanabhan V, Salvetti NR, Matiller V & Ortega HH 2014 Developmental programming: prenatal 476 steroid excess disrupts key members of intraovarian steroidogenic pathway in sheep. Endocrinology 477 155 3649-3660. 478 479 Padmanabhan V & Veiga-Lopez A 2011 Developmental origin of reproductive and metabolic 480 dysfunctions: androgenic versus estrogenic reprogramming. Semin Reprod Med 29 173-186. 481 Padmanabhan V & Veiga-Lopez A 2013 Animal models of the polycystic ovary syndrome phenotype. 482 Steroids 78 734-740. 483 484 Pinilla L, Trimino E, Garnelo P, Bellido C, Aguilar R, Gaytan F & Aguilar E 1993 Changes in pituitary 485 secretion during the early postnatal period and anovulatory syndrome induced by neonatal 486 oestrogen or androgen in rats. J Reprod Fertil 97 13-20. 487 Plant TM & Ramaswamy S 2009 Kisspeptin and the regulation of the hypothalamic-pituitary-gonadal 488 axis in the rhesus monkey (Macaca mulatta). Peptides 30 67-75. 489 490 Recabarreni SE, Sir-Petermann T, Lobos A, Codner E, Rojas-Garcia PP & Reyes V 2005 Response to the 491 gonadotropin releasing hormone agonist leuprolide in immature female sheep androgenized in 492 utero. Biol Res 38 235-244. 493
Rigo ML, Dau AMP, Glanzner WG, Martins M, Zanella R, Rizzetti TM, Comim FV & Gonçalves PBD 494 2015 Steroidogenic enzymes mRNA expression profile and steroids production in bovine theca cells 495 cultured in vitro and stimulated with angiotensin II. Ciência Rural 45 704-710. 496 497 Robinson JE, Forsdike RA & Taylor JA 1999 In utero exposure of female lambs to testosterone 498 reduces the sensitivity of the gonadotropin-releasing hormone neuronal network to inhibition by 499 progesterone. Endocrinology 140 5797-5805. 500 501 Roland AV, Nunemaker CS, Keller SR & Moenter SM 2010 Prenatal androgen exposure programs 502 metabolic dysfunction in female mice. J Endocrinol 207 213-223. 503 504 Ronnekleiv OK & Kelly MJ 2013 Kisspeptin excitation of GnRH neurons. Adv Exp Med Biol 784 113-505 131. 506 507 Schirman-Hildesheim TD, Bar T, Ben-Aroya N & Koch Y 2005 Differential gonadotropin-releasing 508 hormone (GnRH) and GnRH receptor messenger ribonucleic acid expression patterns in different 509 tissues of the female rat across the estrous cycle. Endocrinology 146 3401-3408. 510 511 Singh P & Krishna A 2010 Effects of GnRH agonist treatment on steroidogenesis and folliculogenesis 512 in the ovary of cyclic mice. J Ovarian Res 3 26. 513 514 Skorupskaite K, George JT & Anderson RA 2014 The kisspeptin-GnRH pathway in human reproductive 515 health and disease. Hum Reprod Update 20 485-500. 516 517 Slob AK, den Hamer R, Woutersen PJ & van der Werff ten Bosch JJ 1983 Prenatal testosterone 518 propionate and postnatal ovarian activity in the rat. Acta Endocrinol (Copenh) 103 420-427. 519 520 Sullivan SD & Moenter SM 2005 GABAergic integration of progesterone and androgen feedback to 521 gonadotropin-releasing hormone neurons. Biol Reprod 72 33-41. 522 523 Swanson HE & Werfftenbosch JJ 1964 The "Early-Androgen" Syndrome; Differences in Response to 524 Pre-Natal and Post-Natal Administration of Various Doses of Testosterone Propionate in Female and 525 Male Rats. Acta Endocrinol (Copenh) 47 37-50. 526 527 Tehrani FR, Noroozzadeh M, Zahediasl S, Piryaei A & Azizi F 2014 Introducing a rat model of prenatal 528 androgen-induced polycystic ovary syndrome in adulthood. Exp Physiol 99 792-801. 529 530 Torrealday S, Lalioti MD, Guzeloglu-Kayisli O & Seli E 2013 Characterization of the gonadotropin 531 releasing hormone receptor (GnRHR) expression and activity in the female mouse ovary. 532 Endocrinology 154 3877-3887. 533 534 Tyndall V, Broyde M, Sharpe R, Welsh M, Drake AJ & McNeilly AS 2012 Effect of androgen treatment 535 during foetal and/or neonatal life on ovarian function in prepubertal and adult rats. Reproduction 536 143 21-33. 537 538 Unsworth WP, Taylor JA & Robinson JE 2005 Prenatal programming of reproductive neuroendocrine 539 function: the effect of prenatal androgens on the development of estrogen positive feedback and 540 ovarian cycles in the ewe. Biol Reprod 72 619-627. 541 542 Veiga-Lopez A, Astapova OI, Aizenberg EF, Lee JS & Padmanabhan V 2009 Developmental 543 programming: contribution of prenatal androgen and estrogen to estradiol feedback systems and 544 periovulatory hormonal dynamics in sheep. Biol Reprod 80 718-725. 545
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546 Walters KA, Allan CM & Handelsman DJ 2008 Androgen actions and the ovary. Biol Reprod 78 380-547 389. 548 549 Walters KA, Allan CM & Handelsman DJ 2012 Rodent models for human polycystic ovary syndrome. 550 Biol Reprod 86 149, 141-112. 551 552 Weisz J & Lloyd CW 1965 Estrogen and androgen production in vitro from 7-3-H-progesterone by 553 normal and polycystic rat ovaries. Endocrinology 77 735-744. 554 555 West C, Foster DL, Evans NP, Robinson J & Padmanabhan V 2001 Intra-follicular activin availability is 556 altered in prenatally-androgenized lambs. Mol Cell Endocrinol 185 51-59. 557 558 Wilkinson M & Moger WH 1981 Development of a quotidian increase in pituitary responsiveness to 559 GnRH in prepubertal female rats. J Reprod Fertil 62 393-398. 560 561 Wu XY, Li ZL, Wu CY, Liu YM, Lin H, Wang SH & Xiao WF 2010 Endocrine traits of polycystic ovary 562 syndrome in prenatally androgenized female Sprague-Dawley rats. Endocr J 57 201-209. 563 564 Zurvarra FM, Salvetti NR, Mason JI, Velazquez MM, Alfaro NS & Ortega HH 2009 Disruption in the 565 expression and immunolocalisation of steroid receptors and steroidogenic enzymes in letrozole-566 induced polycystic ovaries in rat. Reprod Fertil Dev 21 827-839. 567
568
Figure 1. Timeline of treatments 569
570
Figure 2. Proportion of estrus cycles in female rats 571
The proportion of each one of the stages of the cycle, as defined by vaginal cytology is presented for 572
each group. Lupron treatment markedly improved estrus in PostN rats. In PreN rats, the 573
administration of leuprolide led to an increase in the estrus cycles (* = p <0.05 treated versus non-574
treated subgroups). Supplemental figure S1- S6 includes individual information on each animal. 575
576
Figure 3. Follicle development 577
Key ovarian structures (corpus luteum, cystic/atretic follicles, and healthy antral follicles) were 578
compared among the three main groups (Control, PostN, and PreN) (Fig D, H and L) and between 579
each subgroup regarding leuprolide acetate treatment (A-C; E-G; I-K). A significant reduction in the 580
number of corpus luteum in the PostN group (Fig 3D; p=0.001) and an increase in cysts/atretic 581
follicles (Fig 3H; p=0.0004) were observed in the PostN group in relation to other groups (Control and 582
PreN). The treatment with leuprolide significantly reduced the number of atretic/cystic follicles 583
(PostN L) (Fig 3G; p=0.04); there was a trend to increase corpus luteum in the same group (Fig 3C). 584
Control animals that were treated (Control L) experienced a trend to reduce healthy antral follicles 585
(Fig 3J). 586
587
588
Figure 4. Testosterone and androstenedione serum levels 589
Levels of testosterone (Fig 4D) and androstenedione (Fig 4H) in serum measured by liquid 590
chromatography (MS/MS) were compared among the three main groups (Control, PostN, and PreN) 591
and between each subgroup regarding leuprolide acetate treatment (A-C; E-G). The PostN L group 592
exhibited a significant reduction in testosterone after treatment with leuprolide acetate (Fig 4B; 593
p=0.04). 594
595
Figure 5. Expression of steroidogenic enzymes and Gnrhr in the ovary 596
A comparison of mRNA expression of CYP11a1, CYP17a1, CYP19a1, and Gnrhr was performed among 597
the three main groups (Control, PostN, and PreN) (Fig 5D, H, L, and P) and between each subgroup 598
regarding leuprolide acetate treatment (A-C; E-G; I-K; M-O). A significantly higher expression of 599
CYP17a1 (Fig 5H; p=0.016) and Gnrhr (Fig 5P; p=0.003) was observed in the PreN group. PreN L 600
animals exhibited a marked increase in Gnrhr expression versus PreN (Fig 5N; p=0.04). 601
602
Figure 6. Expression of Kiss, Gnrh, and Gnrhr in the hypothalamus 603 A comparison of mRNA expression of Kiss, Gnrh, and Gnrhr was performed among the three main 604 groups (Control, PostN, and PreN) (Fig 5D, H, and L) and between each subgroup regarding leuprolide 605 acetate treatment (A-C; E-G; I-K). PostN animals exhibited a higher expression of Gnrhr than Control 606 or PreN rats (Fig 6H; p= 0.001). PreN group demonstrated a significantly higher expression of Gnrh 607 (Fig 6L; p=0.01) and lower Kiss (Fig 6D; p=0.01). Treatment with leuprolide increased Kiss in this 608 group, as well as Gnrh in 609 610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
53
Figure 1. Timeline of treatments. 625
626
627
628
Figure 2. Proportion of estrus cycles in female rats. 629
630
631
632
633
634
635
636
55
Figure 3. Follicle development. 637
638
639
640
641
Figure 4. Testosterone and androstenedione serum levels 642
643
644
645
646
647
648
57
Figure 5. Expression of steroidogenic enzymes and GNRHR in the ovary. 649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
Figure 6. Expression of Kiss, Gnrh, and Gnrhr in the hypothalamus.
59
Supplementary 1.Estrus cycles in female rats PreN. 669
670
671
Supplementary 2. Estrus cycles in female rats PreN L. 672
673
674
61
675
Supplementary 3. Estrus cycles in female rats PosN. 676
677
678
679
Supplementary 4. Estrus cycles in female rats PosN L. 680
681
63
682
683
Supplementary 5. Estrus cycles in female rats Control L 684
685
Supplementary 6. Estrus cycles in female rats PreN Control. 686
687
688
689
65
690
691
Supplementary 7.Estrus cycles in female rats PostN Control. 692
693
694
Supplementary 8. List of primers. 695
Gene Forward primer (5’-3’) Reverse primer (5’-3’)
marcadas na morfologia ovariana (redução de corpo lúteo e aumento de cistos/folículos
atréticos) sem achados de hiperandrogenismo.
De acordo com as evidências obtidas, pode-se sugerir uma participação do eixo do
GnRH no desenvolvimento destas alterações reprodutivas, uma vez que o bloqueio dos
receptores do GnRH pelo uso de acetato de leuprolide por um período aproximado de 4
semanas evitou ou reduziu o desenvolvimento de características de SOP em animais
androgenizados após a gestação (PostN L). No recente estudo de (VEIGA-LOPEZ et al.,
2016) o tratamento pós-natal com flutamida, iniciando as 8 semanas de vida em ovelhas
androgenizadas na gestação foi capaz de melhorar as taxas de androgênio folicular durante a
realização de protocolo de estimulação ovariana. Similarmente, em modelo pós-natal com
roedores, o uso de metformina mostrou benefício na redução de folículos atréticos
(MAHAMED et al., 2011).
No elegante trabalho de (TYNDALL et al., 2012), os tratamentos com testosterona
propianato em diferentes esquemas - fetal (dia e14- e21 de gestação) ou fetal e pós-natal
tardio (incluindo o dia 15º ao 24ºdias de vida), ou ainda fetal e pós-natal pleno (incluindo o
dia 1º ao 24ºdias de vida) não foram capazes de levar às alterações fenotípicas características
de SOP identificadas somente no modelo pós-natal pleno (dia 1º ao 24ºdias de vida). Isto
sugere a possibilidade de existência de alguma condição ou janela na linha do tempo para o
desenvolvimento de características distintas da SOP. Embora a foliculogênese esteja
completamente concluída em mulheres, nos demais primatas e em ovelhas antes do final da
gestação, o mesmo não ocorre com roedores, onde este processo de formação folicular só
encerra nos primeiros dias de vida. Curiosamente, nos primeiros 30 dias de vida em ratas
ocorre um período fisiológico de grande ativação do eixo GnRH (que corresponderia à mini-
puberdade de humanos). O significado da minipuberdade no desenvolvimento de SOP ainda
é incerto.
Evidência na literatura tem sugerido que o excesso de T pré-natal pode reprogramar a
secreção hipotalâmica de GnRH e sua rede(PADMANABHAN & VEIGA-LOPEZ, 2011).
Níveis excessivos de androgênios no período pré-natal em ratas e camundongas geralmente
produz níveis mais elevados de LH associados a um aumento da transmissão GABAérgica
(SULLIVAN & MOENTER, 2005; ROLAND et al., 2010). Além disto, sabe-se que existe
em roedores, um dimorfismo sexual para neurônios do GnRH(MCNEILLY et al., 2003); o
tratamento pré-natal (a maioria das vezes com DHT ou T em níveis mais elevados) leva ao
aumento dos níveis “masculinos” de LH (mas não de FSH) o que pode afetar a ovulação e
produção de androgênios (MCNEILLY et al., 2003; SULLIVAN & MOENTER, 2005;
ROLAND et al., 2010) ). Ademais, níveis de RNAm de kisspeptina foram elevados no
hipotálamo de camundongas tratadas com letrazole (inibidor da aromatase) que está
associado a diversas alterações de ciclicidade e alteração dos níveis de GnRH (KAUFFMAN
et al., 2015)
Em ovelhas, a androgenização pré-natal pode levar a alterações dos neurônios
hipotalâmicos KNDy responsáveis pela modulação da pulsatilidade da secreção de GnRH,
quer seja pela diminuição da neurocinina B, dimorfina ou pela manutenção da expressão de
kisspeptina(CHENG et al., 2010). Apenas o emprego de DHEA e inibidores de aromatase
(como letrozole) após o nascimento rompeu os ciclos ovulatórios e aumento dos níveis de
LH (ZURVARRA et al., 2009).
69
4. CONCLUSÃO
Os resultados do estudo indicam que a androgenização com em ratas Wistar leva a
diferentes mudanças reprodutivas de acordo com o período de emprego do propianato de
testosterona. Comparativamente, o modelo pós-natal (PosN) reproduziu características
importantes da SOP como anovulação e aumento de cistos/folículos atréticos (mas não
hiperandrogenemia) após os 90 dias de vida. Com efeito, o tratamento neonatal com acetato
de leuprolide mostrou-se capaz de impedir o desenvolvimento deste fenótipo (grupo PosN L),
reduzindo os níveis de androgênios, número de cistos/folículos atrésicos. Em nosso estudo
(gerador de hipóteses), a minipuberdade (que termina com a supressão do eixo gonadal)
poderia ser considerada uma janela para estudo e possivelmente intervenções terapêuticas
para a síndrome dos ovários policísticos.
REFERÉNCIA
ABBOTT, D. H., et al. Androgen excess fetal programming of female reproduction: a developmental aetiology for polycystic ovary syndrome? Hum Reprod Update, v.11, n.4, p.357-74. 2005. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/15941725>. Acesso em: 15 abr. 2011. doi: 10.1093/humupd/dmi013.
ABBOTT, D. H., et al. Nonhuman primate models of polycystic ovary syndrome. Mol Cell Endocrinol, v.373, n.1-2, p.21-8. 2013. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/23370180>. Acesso em: 15 abr. 2011. doi: 10.1016/j.mce.2013.01.013.
ABBOTT, D. H., et al. Contributions of androgen and estrogen to fetal programming of ovarian dysfunction. Reprod Biol Endocrinol, v.4, p.17. 2006. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/16606451>. Acesso em: 15 abr. 2011. doi: 10.1186/1477-7827-4-17.
ABBOTT, D. H., et al. Fetal programming of adrenal androgen excess: lessons from a nonhuman primate model of polycystic ovary syndrome. Endocr Dev, v.13, p.145-58. 2008. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/18493139>. Acesso em: 15 abr. 2011. doi: 10.1159/000134831.
ABI SALLOUM, B., et al. Developmental programming: impact of prenatal exposure to bisphenol-A and methoxychlor on steroid feedbacks in sheep. Toxicol Appl Pharmacol, v.268, n.3, p.300-8. 2013. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/23454450>. Acesso em: 15 abr. 2011. doi: 10.1016/j.taap.2013.02.011.
ALEXANDERSON, C., et al. Postnatal testosterone exposure results in insulin resistance, enlarged mesenteric adipocytes, and an atherogenic lipid profile in adult female rats: comparisons with estradiol and dihydrotestosterone. Endocrinology, v.148, n.11, p.5369-76. 2007. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/17656458>. Acesso em: 15 abr. 2011. doi: 10.1210/en.2007-0305.
AYOOLA, A. B., et al. Women's Knowledge of Ovulation, the Menstrual Cycle, and Its Associated Reproductive Changes. Birth. 2016. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/27157718>. Acesso em: 15 abr. 2011. doi: 10.1111/birt.12237.
AZZIZ, R., et al. The Androgen Excess and PCOS Society criteria for the polycystic ovary syndrome: the complete task force report. Fertil Steril, v.91, n.2, p.456-88. 2009. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/18950759>. Acesso em: 15 abr. 2011. doi: 10.1016/j.fertnstert.2008.06.035.
71
AZZIZ, R., et al. The prevalence and features of the polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab, v.89, n.6, p.2745-9. 2004. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/15181052>. Acesso em: 15 abr. 2011. doi: 10.1210/jc.2003-032046.
BARRACLOUGH, C. A. Production of anovulatory, sterile rats by single injections of testosterone propionate. Endocrinology, v.68, p.62-7. 1961. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/13687241>. Acesso em: 15 abr. 2011. doi: 10.1210/endo-68-1-62.
BERNARD, C. An introduction to the study of experimental medicine. 1965
BILLIAR, R. B., et al. The effect of chronic and acyclic elevation of circulating androstenedione or estrone concentrations on ovarian function in the rhesus monkey. Endocrinology, v.116, n.6, p.2209-20. 1985. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/3922743>. Acesso em: 15 abr. 2011. doi: 10.1210/endo-116-6-2209.
BRIDGES, N. A., et al. Standards for ovarian volume in childhood and puberty. Fertil Steril, v.60, n.3, p.456-60. 1993. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/8375526>. Acesso em: 15 abr. 2011. doi.
CALDWELL, A. S., et al. Characterization of reproductive, metabolic, and endocrine features of polycystic ovary syndrome in female hyperandrogenic mouse models. Endocrinology, v.155, n.8, p.3146-59. 2014. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/24877633>. Acesso em: 15 abr. 2011. doi: 10.1210/en.2014-1196.
CARMINA, E., et al. Phenotypic variation in hyperandrogenic women influences the findings of abnormal metabolic and cardiovascular risk parameters. J Clin Endocrinol Metab, v.90, n.5, p.2545-9. 2005. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/15728203>. Acesso em: 15 abr. 2011. doi: 10.1210/jc.2004-2279.
CHEN, X., et al. Prevalence of polycystic ovary syndrome in unselected women from southern China. Eur J Obstet Gynecol Reprod Biol, v.139, n.1, p.59-64. 2008. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/18378061>. Acesso em: 15 abr. 2011. doi: 10.1016/j.ejogrb.2007.12.018.
CHENG, G., et al. The kisspeptin/neurokinin B/dynorphin (KNDy) cell population of the arcuate nucleus: sex differences and effects of prenatal testosterone in sheep. Endocrinology, v.151, n.1, p.301-11. 2010. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/19880810>. Acesso em: 15 abr. 2011. doi: 10.1210/en.2009-0541.
DASGUPTA, S.; B. M. REDDY. Present status of understanding on the genetic etiology of polycystic ovary syndrome. J Postgrad Med, v.54, n.2, p.115-25. 2008. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/18480528>. Acesso em: 15 abr. 2011. doi.
DEVOTO, L., et al. Human corpus luteum physiology and the luteal-phase dysfunction associated with ovarian stimulation. Reprod Biomed Online, v.18 Suppl 2, p.19-24. 2009. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/19406027>. Acesso em: 15 abr. 2011. doi.
DIAMANTI-KANDARAKIS, E., et al. Endocrine-disrupting chemicals: an Endocrine Society scientific statement. Endocr Rev, v.30, n.4, p.293-342. 2009. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/19502515>. Acesso em: 15 abr. 2011. doi: 10.1210/er.2009-0002.
DIAMANTI-KANDARAKIS, E., et al. A survey of the polycystic ovary syndrome in the Greek island of Lesbos: hormonal and metabolic profile. J Clin Endocrinol Metab, v.84, n.11, p.4006-11. 1999. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/10566641>. Acesso em: 15 abr. 2011. doi: 10.1210/jcem.84.11.6148.
DIAMANTI-KANDARAKIS, E., et al. Polycystic ovary syndrome: the influence of environmental and genetic factors. Hormones (Athens), v.5, n.1, p.17-34. 2006. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/16728382>. Acesso em: 15 abr. 2011. doi.
DUMESIC, D. A., et al. Polycystic ovary syndrome and its developmental origins. Rev Endocr Metab Disord, v.8, n.2, p.127-41. 2007. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/17659447>. Acesso em: 15 abr. 2011. doi: 10.1007/s11154-007-9046-0.
DUMESIC, D. A., et al. Early origins of polycystic ovary syndrome. Reprod Fertil Dev, v.17, n.3, p.349-60. 2005. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/15745643>. Acesso em: 15 abr. 2011. doi.
DUMESIC, D. A., et al. Impaired developmental competence of oocytes in adult prenatally androgenized female rhesus monkeys undergoing gonadotropin stimulation for in vitro fertilization. J Clin Endocrinol Metab, v.87, n.3, p.1111-9. 2002. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/11889174>. Acesso em: 15 abr. 2011. doi: 10.1210/jcem.87.3.8287.
DUNAIF, A. Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr Rev, v.18, n.6, p.774-800. 1997. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/9408743>. Acesso em: 15 abr. 2011. doi: 10.1210/edrv.18.6.0318.
EISNER, J. R., et al. Ovarian hyperandrogenism in adult female rhesus monkeys exposed to prenatal androgen excess. Fertil Steril, v.77, n.1, p.167-72. 2002. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/11779609>. Acesso em: 15 abr. 2011. doi.
ERICKSON, G. F. An analysis of follicle development and ovum maturation In: Seminars and reproductive endocrinology. San Diego, California. 1986. 233 - 254 p.
73
ESCOBAR-MORREALE, H. F., et al. The molecular-genetic basis of functional hyperandrogenism and the polycystic ovary syndrome. Endocr Rev, v.26, n.2, p.251-82. 2005. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/15561799>. Acesso em: 15 abr. 2011. doi: 10.1210/er.2004-0004.
FOECKING, E. M., et al. Neuroendocrine consequences of prenatal androgen exposure in the female rat: absence of luteinizing hormone surges, suppression of progesterone receptor gene expression, and acceleration of the gonadotropin-releasing hormone pulse generator. Biol Reprod, v.72, n.6, p.1475-83. 2005. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/15744016>. Acesso em: 15 abr. 2011. doi: 10.1095/biolreprod.105.039800.
FORTUNE, J. E. Ovarian follicular growth and development in mammals. Biol Reprod, v.50, n.2, p.225-32. 1994. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/8142540>. Acesso em: 15 abr. 2011. doi.
FOSTER, D. L. Luteinizing hormone and progesterone secretion during sexual maturation of the rhesus monkey: short luteal phases during the initial menstrual cycles. Biol Reprod, v.17, n.4, p.584-90. 1977. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/411527>. Acesso em: 15 abr. 2011. doi.
FRANKS, S. Animal models and the developmental origins of polycystic ovary syndrome: increasing evidence for the role of androgens in programming reproductive and metabolic dysfunction. Endocrinology, v.153, n.6, p.2536-8. 2012. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/22610962>. Acesso em: 15 abr. 2011. doi: 10.1210/en.2012-1366.
GAMBINERI, A., et al. Obesity and the polycystic ovary syndrome. Int J Obes Relat Metab Disord, v.26, n.7, p.883-96. 2002. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/12080440>. Acesso em: 15 abr. 2011. doi: 10.1038/sj.ijo.0801994.
GARZO, V. G., et al. Effects of an antiprogesterone (RU486) on the hypothalamic-hypophyseal-ovarian-endometrial axis during the luteal phase of the menstrual cycle. J Clin Endocrinol Metab, v.66, n.3, p.508-17. 1988. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/2832438>. Acesso em: 15 abr. 2011. doi: 10.1210/jcem-66-3-508.
GORE, A. C., et al. Early life exposure to endocrine-disrupting chemicals causes lifelong molecular reprogramming of the hypothalamus and premature reproductive aging. Mol Endocrinol, v.25, n.12, p.2157-68. 2011. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/22016562>. Acesso em: 15 abr. 2011. doi: 10.1210/me.2011-1210.
IWASA, T., et al. Effects of chronic testosterone administration on body weight and food intake differ among pre-pubertal, gonadal-intact, and ovariectomized female rats. Behav Brain Res, v.309, p.35-43. 2016. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/27139935>. Acesso em: 15 abr. 2011. doi: 10.1016/j.bbr.2016.04.048.
KAFALI, H., et al. Letrozole-induced polycystic ovaries in the rat: a new model for cystic ovarian disease. Arch Med Res, v.35, n.2, p.103-8. 2004. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/15010188>. Acesso em: 15 abr. 2011. doi: 10.1016/j.arcmed.2003.10.005.
KAUFFMAN, A. S., et al. A Novel Letrozole Model Recapitulates Both the Reproductive and Metabolic Phenotypes of Polycystic Ovary Syndrome in Female Mice. Biol Reprod, v.93, n.3, p.69. 2015. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/26203175>. Acesso em: 15 abr. 2011. doi: 10.1095/biolreprod.115.131631.
KIERSZENBAUM, A. L. Histology and cell biology: an introduction to pathology. 2008. 445-7 p.
KIM, J. J.; Y. M. CHOI. Dyslipidemia in women with polycystic ovary syndrome. Obstet Gynecol Sci, v.56, n.3, p.137-42. 2013. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/24327994>. Acesso em: 15 abr. 2011. doi: 10.5468/ogs.2013.56.3.137.
KUMAR, A., et al. Prevalence of adrenal androgen excess in patients with the polycystic ovary syndrome (PCOS). Clin Endocrinol (Oxf), v.62, n.6, p.644-9. 2005. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/15943823>. Acesso em: 15 abr. 2011. doi: 10.1111/j.1365-2265.2005.02256.x.
LANG, I. A., et al. Association of urinary bisphenol A concentration with medical disorders and laboratory abnormalities in adults. JAMA, v.300, n.11, p.1303-10. 2008. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/18799442>. Acesso em: 15 abr. 2011. doi: 10.1001/jama.300.11.1303.
LEVI-SETTI, P. E., et al. FSH and LH together in ovarian stimulation. Eur J Obstet Gynecol Reprod Biol, v.115 Suppl 1, p.S34-9. 2004. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/15196714>. Acesso em: 15 abr. 2011. doi: 10.1016/j.ejogrb.2004.01.013.
LYONS, A. Medicine: an illustrated history. New York. 1987
MACIEL, G. A., et al. Stockpiling of transitional and classic primary follicles in ovaries of women with polycystic ovary syndrome. J Clin Endocrinol Metab, v.89, n.11, p.5321-7. 2004. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/15531477>. Acesso em: 15 abr. 2011. doi: 10.1210/jc.2004-0643.
MACHADO, A. Neuroanatomia functional. São Paulo. 2006
75
MAHAMED, R. R., et al. Effects of metformin on the reproductive system of androgenized female rats. Fertil Steril, v.95, n.4, p.1507-9. 2011. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/20828684>. Acesso em: 15 abr. 2011. doi: 10.1016/j.fertnstert.2010.07.1093.
MANIKKAM, M., et al. Fetal programming: prenatal testosterone excess leads to fetal growth retardation and postnatal catch-up growth in sheep. Endocrinology, v.145, n.2, p.790-8. 2004. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/14576190>. Acesso em: 15 abr. 2011. doi: 10.1210/en.2003-0478.
MANIKKAM, M., et al. Developmental programming: impact of prenatal testosterone excess on pre- and postnatal gonadotropin regulation in sheep. Biol Reprod, v.78, n.4, p.648-60. 2008. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/18094361>. Acesso em: 15 abr. 2011. doi: 10.1095/biolreprod.107.063347.
MANNERAS, L., et al. A new rat model exhibiting both ovarian and metabolic characteristics of polycystic ovary syndrome. Endocrinology, v.148, n.8, p.3781-91. 2007. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/17495003>. Acesso em: 15 abr. 2011. doi: 10.1210/en.2007-0168.
MARCONDES, F. K., et al. Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol, v.62, n.4A, p.609-14. 2002. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/12659010>. Acesso em: 15 abr. 2011. doi.
MARCONDES, R. R., et al. Differences in neonatal exposure to estradiol or testosterone on ovarian function and hormonal levels. Gen Comp Endocrinol, v.212, p.28-33. 2015. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/25623143>. Acesso em: 15 abr. 2011. doi: 10.1016/j.ygcen.2015.01.006.
MARCH, W. A., et al. The prevalence of polycystic ovary syndrome in a community sample assessed under contrasting diagnostic criteria. Hum Reprod, v.25, n.2, p.544-51. 2010. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/19910321>. Acesso em: 15 abr. 2011. doi: 10.1093/humrep/dep399.
MCDONALD, P. G.; C. DOUGHTY. Comparison of the effect of neonatal administration of testosterone and dihydrotestosterone in the female rat. J Reprod Fertil, v.30, n.1, p.55-62. 1972. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/5064322>. Acesso em: 15 abr. 2011. doi.
MCNEILLY, A. S., et al. The differential secretion of FSH and LH: regulation through genes, feedback and packaging. Reprod Suppl, v.61, p.463-76. 2003. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/14635955>. Acesso em: 15 abr. 2011. doi.
MEHRABIAN, F., et al. The prevalence of polycystic ovary syndrome in Iranian women based on different diagnostic criteria. Endokrynol Pol, v.62, n.3, p.238-42. 2011. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/21717406>. Acesso em: 15 abr. 2011. doi.
MELZER, D., et al. Urinary bisphenol a concentration and angiography-defined coronary artery stenosis. PLoS One, v.7, n.8, p.e43378. 2012. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/22916252>. Acesso em: 15 abr. 2011. doi: 10.1371/journal.pone.0043378.
MICHELMORE, K. F., et al. Polycystic ovaries and associated clinical and biochemical features in young women. Clin Endocrinol (Oxf), v.51, n.6, p.779-86. 1999. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/10619984>. Acesso em: 15 abr. 2011. doi.
MILLER, W. L.; R. J. AUCHUS. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr Rev, v.32, n.1, p.81-151. 2011. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/21051590>. Acesso em: 15 abr. 2011. doi: 10.1210/er.2010-0013.
MORAN, C., et al. Adrenal androgen excess in hyperandrogenism: relation to age and body mass. Fertil Steril, v.71, n.4, p.671-4. 1999. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/10202877>. Acesso em: 15 abr. 2011. doi.
NEWBOLD, R. R., et al. Long-term adverse effects of neonatal exposure to bisphenol A on the murine female reproductive tract. Reprod Toxicol, v.24, n.2, p.253-8. 2007. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/17804194>. Acesso em: 15 abr. 2011. doi: 10.1016/j.reprotox.2007.07.006.
NORMAN, R. J., et al. Polycystic ovary syndrome. The Lancet, v.370, n.9588, p.685-697. 2007. Disponível em: em: 15 abr. 2011. doi: 10.1016/s0140-6736(07)61345-2.
PADMANABHAN, V., et al. Prenatal testosterone excess programs reproductive and metabolic dysfunction in the female. Mol Cell Endocrinol, v.246, n.1-2, p.165-74. 2006. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/16413112>. Acesso em: 15 abr. 2011. doi: 10.1016/j.mce.2005.11.016.
PADMANABHAN, V.; A. VEIGA-LOPEZ. Developmental origin of reproductive and metabolic dysfunctions: androgenic versus estrogenic reprogramming. Semin Reprod Med, v.29, n.3, p.173-86. 2011. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/21710394>. Acesso em: 15 abr. 2011. doi: 10.1055/s-0031-1275519.
77
PADMANABHAN, V.; A. VEIGA-LOPEZ. Animal models of the polycystic ovary syndrome phenotype. Steroids, v.78, n.8, p.734-40. 2013a. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/23701728>. Acesso em: 15 abr. 2011. doi: 10.1016/j.steroids.2013.05.004.
PINILLA, L., et al. Changes in pituitary secretion during the early postnatal period and anovulatory syndrome induced by neonatal oestrogen or androgen in rats. J Reprod Fertil, v.97, n.1, p.13-20. 1993. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/8464003>. Acesso em: 15 abr. 2011. doi.
PRINS, G. S., et al. Developmental estrogen exposures predispose to prostate carcinogenesis with aging. Reprod Toxicol, v.23, n.3, p.374-82. 2007. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/17123779>. Acesso em: 15 abr. 2011. doi: 10.1016/j.reprotox.2006.10.001.
ROBINSON, J. E., et al. In utero exposure of female lambs to testosterone reduces the sensitivity of the gonadotropin-releasing hormone neuronal network to inhibition by progesterone. Endocrinology, v.140, n.12, p.5797-805. 1999. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/10579346>. Acesso em: 15 abr. 2011. doi: 10.1210/endo.140.12.7205.
ROJAS, J., et al. Physiologic Course of Female Reproductive Function: A Molecular Look into the Prologue of Life. J Pregnancy, v.2015, p.715735. 2015. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/26697222>. Acesso em: 15 abr. 2011. doi: 10.1155/2015/715735.
ROLAND, A. V., et al. Prenatal androgen exposure programs metabolic dysfunction in female mice. J Endocrinol, v.207, n.2, p.213-23. 2010. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/20713501>. Acesso em: 15 abr. 2011. doi: 10.1677/JOE-10-0217.
ROSENFIELD, R. L. Current concepts of polycystic ovary syndrome. Baillieres Clin Obstet Gynaecol, v.11, n.2, p.307-33. 1997. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/9536213>. Acesso em: 15 abr. 2011. doi.
RUSSELL, B. História do pensamento ocidental. São Paulo. 2001
SAM, S.; D. A. EHRMANN. Hormonal Evaluation of Hyperandrogenism in Women. JAMA, v.314, n.23, p.2557-8. 2015. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/26670974>. Acesso em: 15 abr. 2011. doi: 10.1001/jama.2015.11612.
SARMA, H. N., et al. Fetal programming: excess prenatal testosterone reduces postnatal luteinizing hormone, but not follicle-stimulating hormone responsiveness, to estradiol negative feedback in the female. Endocrinology, v.146, n.10, p.4281-91. 2005. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/15976056>. Acesso em: 15 abr. 2011. doi: 10.1210/en.2005-0322.
SHARMA, T. P., et al. Fetal programming: prenatal androgen disrupts positive feedback actions of estradiol but does not affect timing of puberty in female sheep. Biol Reprod, v.66, n.4, p.924-33. 2002. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/11906910>. Acesso em: 15 abr. 2011. doi.
SINGH, K. B. Persistent estrus rat models of polycystic ovary disease: an update. Fertil Steril, v.84 Suppl 2, p.1228-34. 2005. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/16210015>. Acesso em: 15 abr. 2011. doi: 10.1016/j.fertnstert.2005.06.013.
SLOB, A. K., et al. Prenatal testosterone propionate and postnatal ovarian activity in the rat. Acta Endocrinol (Copenh), v.103, n.3, p.420-7. 1983. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/6880571>. Acesso em: 15 abr. 2011. doi.
SPEROFF L, F. M. Clinical Endocrinology and infertility. Philadelphia. 2005
STECKLER, T. L., et al. Developmental programming in sheep: administration of testosterone during 60-90 days of pregnancy reduces breeding success and pregnancy outcome. Theriogenology, v.67, n.3, p.459-67. 2007. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/17010414>. Acesso em: 15 abr. 2011. doi: 10.1016/j.theriogenology.2006.08.010.
STEVENS A, L. J. L. Macrophages and Dendritic Cells. In: Human Histology. 2002. 117-23 p.
SULLIVAN, S. D.; S. M. MOENTER. GABAergic integration of progesterone and androgen feedback to gonadotropin-releasing hormone neurons. Biol Reprod, v.72, n.1, p.33-41. 2005. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/15342358>. Acesso em: 15 abr. 2011. doi: 10.1095/biolreprod.104.033126.
SWANSON, H. E.; J. J. WERFFTENBOSCH. The "Early-Androgen" Syndrome; Differences in Response to Pre-Natal and Post-Natal Administration of Various Doses of Testosterone Propionate in Female and Male Rats. Acta Endocrinol (Copenh), v.47, p.37-50. 1964. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/14208150>. Acesso em: 15 abr. 2011. doi.
79
TARANTINO, G., et al. Bisphenol A in polycystic ovary syndrome and its association with liver-spleen axis. Clin Endocrinol (Oxf), v.78, n.3, p.447-53. 2013. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/22805002>. Acesso em: 15 abr. 2011. doi: 10.1111/j.1365-2265.2012.04500.x.
TEHRANI, F. R., et al. Introducing a rat model of prenatal androgen-induced polycystic ovary syndrome in adulthood. Exp Physiol, v.99, n.5, p.792-801. 2014. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/24532600>. Acesso em: 15 abr. 2011. doi: 10.1113/expphysiol.2014.078055.
TYNDALL, V., et al. Effect of androgen treatment during foetal and/or neonatal life on ovarian function in prepubertal and adult rats. Reproduction, v.143, n.1, p.21-33. 2012. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/22016380>. Acesso em: 15 abr. 2011. doi: 10.1530/REP-11-0239.
UNSWORTH, W. P., et al. Prenatal programming of reproductive neuroendocrine function: the effect of prenatal androgens on the development of estrogen positive feedback and ovarian cycles in the ewe. Biol Reprod, v.72, n.3, p.619-27. 2005. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/15509728>. Acesso em: 15 abr. 2011. doi: 10.1095/biolreprod.104.035691.
VAN DER KAMP, H. J., et al. Newborn screening for congenital adrenal hyperplasia in the Netherlands. Pediatrics, v.108, n.6, p.1320-4. 2001. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/11731654>. Acesso em: 15 abr. 2011. doi.
VEIGA-LOPEZ, A., et al. Developmental programming: rescuing disruptions in preovulatory follicle growth and steroidogenesis from prenatal testosterone disruption. J Ovarian Res, v.9, n.1, p.39. 2016. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/27357284>. Acesso em: 15 abr. 2011. doi: 10.1186/s13048-016-0250-y.
VEIGA-LOPEZ, A., et al. Developmental programming: deficits in reproductive hormone dynamics and ovulatory outcomes in prenatal, testosterone-treated sheep. Biol Reprod, v.78, n.4, p.636-47. 2008. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/18094354>. Acesso em: 15 abr. 2011. doi: 10.1095/biolreprod.107.065904.
VIEIRA S, H. W. A ética e a metodologia. 1998
WALTERS, K. A., et al. Androgen actions and the ovary. Biol Reprod, v.78, n.3, p.380-9. 2008. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/18003945>. Acesso em: 15 abr. 2011. doi: 10.1095/biolreprod.107.064089.
WEST, C., et al. Intra-follicular activin availability is altered in prenatally-androgenized lambs. Mol Cell Endocrinol, v.185, n.1-2, p.51-9. 2001. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/11738794>. Acesso em: 15 abr. 2011. doi.
WILD, R. A. Dyslipidemia in PCOS. Steroids, v.77, n.4, p.295-9. 2012. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/22197663>. Acesso em: 15 abr. 2011. doi: 10.1016/j.steroids.2011.12.002.
WILSON, M. E.; T. P. GORDON. Season determines timing of first ovulation in rhesus monkeys (Macaca mulatta) housed outdoors. J Reprod Fertil, v.85, n.2, p.583-91. 1989. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/2703996>. Acesso em: 15 abr. 2011. doi.
XU, X. J., et al. Prenatal hyperandrogenic environment induced autistic-like behavior in rat offspring. Physiol Behav, v.138, p.13-20. 2015. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/25455866>. Acesso em: 15 abr. 2011. doi: 10.1016/j.physbeh.2014.09.014.
YEN, J. Reproductive Endocrinology; Physiology, pathophysiology, and clinical management. Pennaylvania. 2004. 3-74 p.
YEN, S. S. Regulation of the hypothalamic--pituitary--ovarian axis in women. J Reprod Fertil, v.51, n.1, p.181-91. 1977. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/335059>. Acesso em: 15 abr. 2011. doi.
ZHOU, R., et al. Adrenal hyperandrogenism is induced by fetal androgen excess in a rhesus monkey model of polycystic ovary syndrome. J Clin Endocrinol Metab, v.90, n.12, p.6630-7. 2005. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/16174719>. Acesso em: 15 abr. 2011. doi: 10.1210/jc.2005-0691.
ZURVARRA, F. M., et al. Disruption in the expression and immunolocalisation of steroid receptors and steroidogenic enzymes in letrozole-induced polycystic ovaries in rat. Reprod Fertil Dev, v.21, n.7, p.827-39. 2009. Disponível em: <http://www.ncbi.nlm.nih.gov/pubmed/19698287>. Acesso em: 15 abr. 2011. doi: 10.1071/RD09026.