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Fetal or neonatal androgen exposure has a programming effect on ovarian function inducing a polycystic ovarian syndrome-like condition. Its effects on uterine structure and function are poorly studied. The aim of this work was to characterize the temporal course of changes in the rat uterine structure induced by neonatal exposure to aromatizable or not aromatizable androgens. Rats were daily treated with testosterone, dihydrotestosterone or vehicle during follicle assembly period (postnatal days 1 to 5). Uterine histoarchitecture, hormonal milieu, endometrial stromal collagen and capillary density were analyzed at prepubertal, pubertal and adult ages. Our data shows that neonatal androgen exposure induces early and long-lasting deleterious effects on uterine development, including altered adenogenesis and superficial epithelial alterations and suggest a role for altered serum estradiol levels in the maintenance and worsening of the situation. Our results suggest that alterations of the neonatal androgenic environment on the uterus could be responsible for alterations in the processes of implantation and maintenance of the embryo in women with polycystic ovary syndrome., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Ovarian surface epithelium (OSE) is a cell monolayer surrounding the ovary; it is involved in the regulation of the ovulatory process and the genesis of ovarian carcinoma. However, intercellular messengers regulating signaling events, like proliferation in the OSE, have not been completely described. Purines have emerged as novel intercellular messengers in the ovary, in which expression of purinergic receptors has been reported in different cell types. In the present work, we described the functional expression of P2Y2 receptor (P2Y2R), a purinergic receptor widely associated with cell proliferation, in the OSE. The expression of P2Y2R by immunofluorescence and RT-PCR, and its functionality by Ca 2+ recording was demonstrated in primary cultured OSE. Functional expression of P2Y2R was also exhibited in situ, by recording of intracellular Ca 2+ release and detection of ERK phosphorylation after injection of a selective agonist into the ovarian bursa. Furthermore, P2Y2R activation with UTPγS, in situ, induced cell proliferation at 24 h, whereas continuous stimulation of P2Y2R during a complete estrous cycle significantly modified the size distribution of the follicular population. This is the first evidence of the functional expression of purinergic P2Y2R in the OSE and opens new perspectives on the roles played by purines in ovarian physiology., (© 2021 Wiley Periodicals LLC.)

Androgens are relevant in order to achieve a normal growth and maturation of the follicle and oocyte, since both excess and absence of androgens may affect the correct ovarian function. The current study analyzes the impact of neonatal androgenization in the first ovulation and oocyte maturation in response to exogenous gonadotrophin stimulation. Neonatal rats were daily treated with testosterone, dihydrotestosterone, or vehicle during follicle assembly period (days 1 to 5). At juvenile period, rats were stimulated sequentially with PMSG and hCG. Ovulation, ovarian histology, hormonal milieu, morphological characteristics of meiotic spindle, and in vitro fertilization rate in oocytes were analyzed. Our data shows that oocytes from androgenized rats displayed a major proportion of aberrant spindles and altered meiotic advance that control animals. These alterations were accompanied with an increase in both fertilization rate and aberrant embryos after 48 h of culture. Our findings showed a direct impact of neonatal androgens on oocyte development; their effects may be recognized at adulthood, supporting the idea of a programming effect exerted by neonatal androgens. These results could be relevant to explain the low fertility rate seen in polycystic ovary syndrome patients after in vitro fertilization procedures., (© 2021. Society for Reproductive Investigation.)

Extracellular purines through specific receptors have been recognized as new regulators of ovarian function. It is known that P2Y2 receptor activity induces theca cell proliferation, we hypothesized that purinergic signaling controls the changes related to hyperthecosis in polycystic ovarian syndrome (PCOS). The aim of this study was to analyze the expression of UTP-sensitive P2Y receptors and their role in theca cells (TC) proliferation in experimentally-induced PCOS (EI-PCOS). In primary cultures of TC from intact rats, all the transcripts of P2Y receptors were detected by polymerase chain reaction; in these cells, UTP (10 μM) induced extracellular signal-regulated kinases (ERK) phosphorylation. Rats with EI-PCOS showed a reduced expression of P2Y2R in TC whereas P2Y4R did not change. By analyzing ERK phosphorylation, it was determined that P2Y2R is the most relevant receptor in TC. UTP promoted cell proliferation in TC from control but not from EI-PCOS rats. The in silico analysis of P2yr2 promoter indicated the presence of androgen response elements; the stimulation of TC primary cultures with testosterone promoted a significant reduction in the expression of the P2yr2 transcript. We concluded that P2Y2R participates in controlling the proliferative rate of TCs from healthy ovaries, but this regulation is lost during EI-PCOS., (© 2019 Wiley Periodicals, Inc.)

This study analyzes the effects of neonatal androgenization on follicular growth and first ovulation in response to gonadotrophins, using a model of exogenous stimulation or the use of subcutaneous ovary grafts in castrated animals to replace the hypothalamus-pituitary signal. Neonatal rats (days 1-5) were treated with testosterone, dihydrotestosterone or vehicle. At juvenile period, rats were stimulated with PMSG, hCG (alone or combined) or used as ovarian donors to be grafted on castrated adult female rats. Ovulation and ovarian histology were analyzed in both groups. Animals treated with vehicle or dihydrotestosterone stimulated with gonadotrophins (pharmacological or by using an ovary graft) ovulated, showing a normal histological morphology whereas rats exposed to testosterone and injected with the same doses of gonadotrophins did not it. In this group, ovulation was reached using a higher dose of hCG. Ovaries in the testosterone group were characterized by the presence of follicles with atretic appearance and a larger size than those observed in control or dihydrotestosterone groups. A similar appearance was observed in testosterone ovary grafts although luteinization and some corpora lutea were also identified. Our findings suggest that neonatal exposure to aromatizable androgens induces a more drastic signalling on the ovarian tissue that those driven by non-aromatizable androgens in response to gonadotrophins.

The effects of neonatal testosterone or dihydrotestosterone exposure on ovarian follicular dynamics were analysed at prepubertal, pubertal or adult age in Wistar rats. Both androgens induced a transitory increase on follicular endowment that was partially corrected at puberty. At adult age testosterone prevented ovulation, without significant modifications on follicular dynamics. An increased number of cystic structures were observed from puberty to adult age. However, ovaries of rats treated with dihydrotestosterone showed follicles with evident morphological alterations in granulosa and thecal layers although several corpora lutea were observed. A significant increase in preantral follicles and few cystic structures were detected at advanced adulthood. The size of cyst increased with age. No immunohistochemical changes on growth factors or enzymes related to steroidogenesis in growing follicles were obvious in any group. In both androgenized groups, cysts shared immunohistochemical characteristics exhibited by preovulatory follicles but they were unable to ovulate spontaneously. Our results provide an insight into the role of different androgens in female reproductive system development, indicating a direct effect of dihydrotestosterone on ovarian tissues whereas a central effect would be the main feature of neonatal testosterone exposure. Heterogeneous clinical manifestations seen in pathologies such as polycystic ovary syndrome among women could be associated with subtle hormonal changes during follicular population development.

This study analysed the temporal association between ovarian cyst development induced by neonatal androgenisation and sympathetic innervation. Neonatal rats (postnatal Days 1 to 5) were treated with testosterone or dihydrotestosterone and the effects were evaluated at postnatal Days 20, 40, 90 or 180. Ovulation rate, number of cystic follicles and density of sympathetic fibres were analysed. The effects of surgical denervation or gonadotrophin stimulation were also assessed. Rats exposed to testosterone showed no oestrous cycle activity and did not ovulate, maintaining a polycystic ovarian morphology at all ages studied. Also, a significant increase in ovarian density of noradrenergic fibres was detected at postnatal Days 90 and 180. Sympathectomy was unable to re-establish ovarian activity; however, human chorionic gonadotrophin stimulation was enough to induce ovulation. The impact of dihydrotestosterone on ovarian function was less noticeable, showing the coexistence of corpora lutea and cystic structures without changes in sympathetic innervation. Our findings suggest that a remodelling of ovarian sympathetic innervation occurs as a response to modifications in the pattern of follicular growth induced by testosterone. A role of sympathetic innervation in the maintenance of the polycystic condition is suggested.

Estradiol is a key hormone in the regulation of reproductive processes acting both on peripheral organs and sympathetic neurons associated to reproductive function. However, many of its regulatory effects on the development and function on the sympathetic neurons have not been completely clarified. Sympathetic neurons located in the celiac ganglion projects to visceral, vascular and glandular targets, and contribute to ovarian innervation, being the main source of sympathetic fibers. In the present study, we analyze the effects of elevated levels of exogenous estrogen during the prepubertal period in post-ganglionic sympathetic neurons. Estrogen exposure induced a significant increase in sympathetic celiac neuronal size and modified the expression of neurotrophin receptor p75. This change affected mainly small and medium size neurons. The effect of estrogens on innervation of celiac target organs was heterogeneous, inducing a significant increase in catecholaminergic innervation of the ovary, but not of the pyloric muscular layers. These findings further support the role of estrogen as a modulator of neuronal plasticity and suggest that estrogen could modify some features involved in the relation between sympathetic immature peripheral neurons and their target organs throughout a neurotrophin-dependent mechanism.

The present study analyses the participation of ovarian innervation during reproductive senescence. We use the model of acute peripheral pharmacological sympathetic denervation with guanethidine in young (3 months old), middle-aged (12 months old) or old (18 months old) rats with spontaneous or induced ovulation. Ovarian levels of norepinephrine (NE) were measured by HPLC and the oestrous cycle, the number of ovulating animals and the percentage of atretic follicles were also assessed. Aged animals showed a progressive reduction in ovulatory capacity and an increase in ovarian NE content. Acute denervation increased the percentage of healthy follicles in 12- and 18-month-old rats compared with control adult animals. Combined treatment of denervation plus stimulation with gonadotrophins doubled the number of ova shed in young adult rats and restablished a partial ovulation in 12-month-old rats. The results suggest that ovarian noradrenergic innervation plays a modulator role in ovarian physiology during the ageing ovary process. The action of ovarian noradrenergic innervation seems to be associated with folliculogenesis and the ovarian response to gonadotrophins.

The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.

Previous studies have shown that chronic administration of oestrogen during postnatal rat development dramatically reduces the total content of noradrenaline in the uterine horn, abolishes myometrial noradrenergic innervation and reduces noradrenaline-fluorescence intensity of intrauterine perivascular nerve fibres. In the present study we analysed if this response is due to a direct and selective effect of oestrogen on the uterine noradrenaline-containing sympathetic nerves, using the in oculo transplantation method. Small pieces of myometrium from prepubertal rats were transplanted into the anterior eye chamber of adult ovariectomised host rats. The effect of systemic chronic oestrogen treatment on the reinnervation of the transplants by noradrenaline-containing sympathetic fibres from the superior cervical ganglion was analysed on cryostat tissue sections processed by the glyoxylic acid technique. In addition, the innervation of the host iris was assessed histochemically and biochemically. The histology of the transplants and irises was examined in toluidine blue-stained semithin sections. These studies showed that after 5 wk in oculo, the overall size of the oestrogen-treated transplants was substantially larger than controls, and histology showed that this change was related to an increase in the size and number of smooth muscle cells within the transplant. Chronic oestrogen treatment did not provoke trophic changes in the irideal muscle. Histochemistry showed that control transplants had a rich noradrenergic innervation, associated with both myometrium and blood vessels. Conversely, in oestrogen-treated transplants only occasional fibres were recognised, showing a reduced NA fluorescence intensity. No changes in the pattern and density of innervation or in the total content of noradrenaline of the host irises were detected after chronic exposure to oestrogen. We interpreted these results to indicate that the effects of oestrogen on uterine noradrenaline-containing sympathetic nerves are neither selective or direct, but result from an interaction between sympathetic nerve fibres with the oestradiol-primed uterine tissue. A potential effect of oestrogen on the neurotrophic capacity of the uterus is discussed.