Spatially coordinated dynamic gene transcription in living pituitary tissue.

Transcription at individual genes in single cells is often pulsatile and stochastic. A key question emerges regarding how this behaviour contributes to tissue phenotype, but it has been a challenge to quantitatively analyse this in living cells over time, as opposed to studying snap-shots of gene expression state. We have used imaging of reporter gene expression to track transcription in living pituitary tissue. We integrated live-cell imaging data with statistical modelling for quantitative real-time estimation of the timing of switching between transcriptional states across a whole tissue. Multiple levels of transcription rate were identified, indicating that gene expression is not a simple binary 'on-off' process. Immature tissue displayed shorter durations of high-expressing states than the adult. In adult pituitary tissue, direct cell contacts involving gap junctions allowed local spatial coordination of prolactin gene expression. Our findings identify how heterogeneous transcriptional dynamics of single cells may contribute to overall tissue behaviour.

Role of Estrogen Response Element in the Human Prolactin Gene: Transcriptional Response and Timing.

The use of bacterial artificial chromosome (BAC) reporter constructs in molecular physiology enables the inclusion of large sections of flanking DNA, likely to contain regulatory elements and enhancers regions that contribute to the transcriptional output of a gene. Using BAC recombineering, we have manipulated a 160-kb human prolactin luciferase (hPRL-Luc) BAC construct and mutated the previously defined proximal estrogen response element (ERE) located -1189 bp relative to the transcription start site, to assess its involvement in the estrogen responsiveness of the entire hPRL locus. We found that GH3 cell lines stably expressing Luc under control of the ERE-mutated hPRL promoter (ERE-Mut) displayed a dramatically reduced transcriptional response to 17ß-estradiol (E2) treatment compared with cells expressing Luc from the wild-type (WT) ERE hPRL-Luc promoter (ERE-WT). The -1189 ERE controls not only the response to E2 treatment but also the acute transcriptional response to TNFα, which was abolished in ERE-Mut cells. ERE-WT cells displayed a biphasic transcriptional response after TNFα treatment, the acute phase of which was blocked after treatment with the estrogen receptor antagonist 4-hydroxy-tamoxifen. Unexpectedly, we show the oscillatory characteristics of hPRL promoter activity in individual living cells were unaffected by disruption of this crucial response element, real-time bioluminescence imaging showed that transcription cycles were maintained, with similar cycle lengths, in ERE-WT and ERE-Mut cells. These data suggest the -1189 ERE is the dominant response element involved in the hPRL transcriptional response to both E2 and TNFα and, crucially, that cycles of hPRL promoter activity are independent of estrogen receptor binding.

Binary Switching of Calendar Cells in the Pituitary Defines the Phase of the Circannual Cycle in Mammals.

Persistent free-running circannual (approximately year-long) rhythms have evolved in animals to regulate hormone cycles, drive metabolic rhythms (including hibernation), and time annual reproduction. Recent studies have defined the photoperiodic input to this rhythm, wherein melatonin acts on thyrotroph cells of the pituitary pars tuberalis (PT), leading to seasonal changes in the control of thyroid hormone metabolism in the hypothalamus. However, seasonal rhythms persist in constant conditions in many species in the absence of a changing photoperiod signal, leading to the generation of circannual cycles. It is not known which cells, tissues, and pathways generate these remarkable long-term rhythmic processes. We show that individual PT thyrotrophs can be in one of two binary states reflecting either a long (EYA3(+)) or short (CHGA(+)) photoperiod, with the relative proportion in each state defining the phase of the circannual cycle. We also show that a morphogenic cycle driven by the PT leads to extensive re-modeling of the PT and hypothalamus over the circannual cycle. We propose that the PT may employ a recapitulated developmental pathway to drive changes in morphology of tissues and cells. Our data are consistent with the hypothesis that the circannual timer may reside within the PT thyrotroph and is encoded by a binary switch timing mechanism, which may regulate the generation of circannual neuroendocrine rhythms, leading to dynamic re-modeling of the hypothalamic interface. In summary, the PT-ventral hypothalamus now appears to be a prime structure involved in long-term rhythm generation.

In an Ovine Model of Polycystic Ovary Syndrome (PCOS) Prenatal Androgens Suppress Female Fetal Renal Gluconeogenesis.

Increased maternal androgen exposure during pregnancy programmes a polycystic ovary syndrome (PCOS)-like condition, with metabolic dysfunction, in adult female offspring. Other in utero exposures associated with the development of insulin resistance, such as intrauterine growth restriction and exposure to prenatal glucocorticoids, are associated with altered fetal gluconeogenesis. We therefore aimed to assess the effect of maternal androgenisation on the expression of PEPCK and G6PC in the ovine fetus. Pregnant Scottish Greyface sheep were treated with twice weekly testosterone propionate (TP; 100mg) or vehicle control from day 62 to day 102 of gestation. At day 90 and day 112 fetal plasma and liver and kidney tissue was collected for analysis. PEPCK and G6PC expression were analysed by quantitative RT-PCR, immunohistochemistry and western blotting. PEPCK and G6PC were localised to fetal hepatocytes but maternal androgens had no effect on female or male fetuses. PEPCK and G6PC were also localised to the renal tubules and renal PEPCK (P<0.01) and G6PC (P = 0.057) were lower in females after prenatal androgenisation with no change in male fetuses. These tissue and sex specific observations could not be explained by alterations in fetal insulin or cortisol. The sexual dimorphism may be related to the increase in circulating estrogen (P<0.01) and testosterone (P<0.001) in females but not males. The tissue specific effects may be related to the increased expression of ESR1 (P<0.01) and AR (P<0.05) in the kidney when compared to the fetal liver. After discontinuation of maternal androgenisation female fetal kidney PEPCK expression normalised. These data further highlight the fetal and sexual dimorphic effects of maternal androgenisation, an antecedent to adult disease and the plasticity of fetal development.

The local effects of ovarian diathermy in an ovine model of polycystic ovary syndrome.

In order to develop a medical alternative to surgical ovarian diathermy (OD) in polycystic ovary syndrome (PCOS) more mechanistic information is required about OD. We therefore studied the cellular, molecular and vascular effects of diathermy on the ovary using an established ovine model of PCOS. Pregnant sheep were treated twice weekly with testosterone propionate (100 mg) from day 30-100 gestation. Their female offspring (n = 12) were studied during their second breeding season when the PCOS-like phenotype, with anovulation, is fully manifest. In one group (n = 4) one ovary underwent diathermy and it was collected and compared to the contralateral ovary after 24 hours. In another group a treatment PCOS cohort underwent diathermy (n = 4) and the ovaries were collected and compared to the control PCOS cohort (n = 4) after 5 weeks. Ovarian vascular indices were measured using contrast-enhanced ultrasound and colour Doppler before, immediately after, 24 hours and five weeks after diathermy. Antral follicles were assessed by immunohistochemistry and ovarian stromal gene expression by quantitative RT-PCR 24 hours and 5 weeks after diathermy. Diathermy increased follicular atresia (P<0.05) and reduced antral follicle numbers after 5 weeks (P<0.05). There was an increase in stromal CCL2 expression 24 hours after diathermy (P<0.01) but no alteration in inflammatory indices at 5 weeks. Immediately after diathermy there was increased microbubble transit time in the ovarian microvasculature (P = 0.05) but this was not seen at 24 hours. However 24 hours after diathermy there was a reduction in the stromal Doppler blood flow signal (P<0.05) and an increased ovarian resistance index (P<0.05) both of which persisted at 5 weeks (P<0.01; P<0.05). In the ovine model of PCOS, OD causes a sustained reduction in ovarian stromal blood flow with an increased ovarian artery resistance index associated with atresia of antral follicles.

Transcriptome profiling of granulosa and theca cells during dominant follicle development in the horse.

Several aspects of equine ovarian physiology are unique among domestic species. Moreover, follicular growth patterns are very similar between horses and humans. This study aimed to characterize, for the first time, global gene expression profiles associated with growth and preovulatory (PO) maturation of equine dominant follicles. Granulosa cells (GCs) and theca interna cells (TCs) were harvested from follicles (n = 5) at different stages of an ovulatory wave in mares corresponding to early dominance (ED; diameter ≥22 mm), late dominance (LD; ≥33 mm) and PO stage (34 h after administration of crude equine gonadotropins at LD stage), and separately analyzed on a horse gene expression microarray, followed by validation using quantitative PCR and immunoblotting/immunohistochemistry. Numbers of differentially expressed transcripts (DETs; ≥2-fold; P < 0.05) during the ED-LD and LD-PO transitions were 546 and 2419 in GCs and 5 and 582 in TCs. The most prominent change in GCs was the down-regulation of transcripts associated with cell division during both ED-LD and LD-PO. In addition, DET sets during LD-PO in GCs were enriched for genes involved in cell communication/adhesion, antioxidation/detoxification, immunity/inflammation, and cholesterol biosynthesis. In contrast, the largest change in TCs during the LD-PO transition was an up-regulation of genes involved in immune activation, with other DET sets mapping to GPCR/cAMP signaling, lipid/amino acid metabolism, and cell proliferation/survival and differentiation. In conclusion, distinct expression profiles were identified between growing and PO follicles and, particularly, between GCs and TCs within each stage. Several DETs were identified that have not been associated with follicle development in other species.

Characterization of microRNAs differentially expressed during bovine follicle development.

Several different miRNAs have been proposed to regulate ovarian follicle function; however, very limited information exists on the spatiotemporal patterns of miRNA expression during follicle development. The objective of this study was to identify, using microarray, miRNA profiles associated with growth and regression of dominant-size follicles in the bovine monovular ovary and to characterize their spatiotemporal distribution during development. The follicles were collected from abattoir ovaries and classified as small (4-8  mm) or large (12-17  mm); the latter were further classified as healthy or atretic based on estradiol and CYP19A1 levels. Six pools of small follicles and individual large healthy (n=6) and large atretic (n=5) follicles were analyzed using Exiqon's miRCURY LNA microRNA Array 6th gen, followed by qPCR validation. A total of 17 and 57 sequences were differentially expressed (greater than or equal to twofold; P<0.05) between large healthy and each of small and large atretic follicles respectively. Bovine miRNAs confirmed to be upregulated in large healthy follicles relative to small follicles (bta-miR-144, bta-miR-202, bta-miR-451, bta-miR-652, and bta-miR-873) were further characterized. Three of these miRNAs (bta-miR-144, bta-miR-202, and bta-miR-873) were also downregulated in large atretic follicles relative to large healthy follicles. Within the follicle, these miRNAs were predominantly expressed in mural granulosa cells. Further, body-wide screening revealed that bta-miR-202, but not other miRNAs, was expressed exclusively in the gonads. Finally, a total of 1359 predicted targets of the five miRNAs enriched in large healthy follicles were identified, which mapped to signaling pathways involved in follicular cell proliferation, steroidogenesis, prevention of premature luteinization, and oocyte maturation.

Excess androgens in utero alters fetal testis development.

Prenatal androgenization induces a polycystic ovary syndrome-like phenotype in adult female offspring, which is associated with alterations that can be detected in the fetal ovary, suggesting gestational origins of this condition. We therefore investigated whether increased prenatal androgen exposure also altered testicular development using ovine animal models. Biweekly maternal testosterone propionate (TP; 100 mg) from day 62 to day 70/day 90 of gestation altered male developmental trajectory. In male fetuses serum LH was decreased (P < .01), and testicular STAR, CYP11, and CYP17 abundance were reduced. Coincident with this, basal testicular T synthesis was decreased in vitro (P < .001). Leydig cell distribution was severely perturbed in all testes prenatally exposed to TP (P < .001). To examine the contribution of estrogens, fetuses were injected with TP (20 mg), the potent estrogen agonist, diethylstilbestrol (DES; 20 mg), or vehicle control at day 62 and day 82 and assessed at day 90. The effects of fetal (direct) TP treatment, but not DES, paralleled maternal (indirect) TP exposure, supporting a direct androgen effect. Cessation of maternal androgenization at day 102 returned Leydig cell distribution to normal but increased basal T output, at day 112, demonstrating Leydig cell developmental plasticity. Earlier maternal androgen exposure from day 30 similarly influenced Leydig cell development at day 90 but additionally affected the expression of Sertoli and germ cell markers. We show in this study that increased prenatal androgen exposure alters development and function of Leydig cells at a time when androgen production is paramount for male development. This supports the concept that gestational antecedents associated with polycystic ovary syndrome may have effects on the male fetus.

The pancreas is altered by in utero androgen exposure: implications for clinical conditions such as polycystic ovary syndrome (PCOS).

Using an ovine model of polycystic ovary syndrome (PCOS), (pregnant ewes injected with testosterone propionate (TP) (100 mg twice weekly) from day (d)62 to d102 of d147 gestation (maternal injection - MI-TP)), we previously reported female offspring with normal glucose tolerance but hyperinsulinemia. We therefore examined insulin signalling and pancreatic morphology in these offspring using quantitative (Q) RT-PCR and western blotting. In addition the fetal pancreatic responses to MI-TP, and androgenic and estrogenic contributions to such responses (direct fetal injection (FI) of TP (20 mg) or diethylstilbestrol (DES) (20 mg) at d62 and d82 gestation) were assessed at d90 gestation. Fetal plasma was assayed for insulin, testosterone and estradiol, pancreatic tissue was cultured, and expression of key ß-cell developmental genes was assessed by QRT-PCR. In female d62MI-TP offspring insulin signalling was unaltered but there was a pancreatic phenotype with increased numbers of ß-cells (P<0.05). The fetal pancreas expressed androgen receptors in islets and genes involved in ß-cell development and function (PDX1, IGF1R, INSR and INS) were up-regulated in female fetuses after d62MI-TP treatment (P<0.05-0.01). In addition the d62MI-TP pancreas showed increased insulin secretion under euglycaemic conditions (P<0.05) in vitro. The same effects were not seen in the male fetal pancreas or when MI-TP was started at d30, before the male programming window. As d62MI-TP increased both fetal plasma testosterone (P<0.05) and estradiol concentrations (P<0.05) we assessed the relative contribution of androgens and estrogens. FI-TP (commencing d62) (not FI-DES treatment) caused elevated basal insulin secretion in vitro and the genes altered by d62MI-TP treatment were similarly altered by FI-TP but not FI-DES. In conclusion, androgen over-exposure alters fetal pancreatic development and ß-cell numbers in offspring. These data suggest that that there may be a primary pancreatic phenotype in models of PCOS, and that there may be a distinct male and female pancreas.

Rodent models of polycystic ovary syndrome.

Rodents are clearly valuable models for assessing disruption of fertility. The effects of different steroid treatments at different stages of reproductive life through from fetal to adult have been assessed for effects on fertility, ovarian morphology, hypothalamic-pituitary function or metabolic consequences. The results show that steroid treatments do disrupt fertility in many cases, but the underlying mechanisms are complicated by the effects of the different treatments at multiple sites. As models for PCOS at the ovarian level however, there are a number of problems particularly related to the fact that rodents are multi-ovular species. Apart from an absence of ovulation and corpora lutea, many of the different steroid regimes result in an increase in large atretic, or cystic follicles that do not parallel PCOS in women. Indeed a number of treatments are given at times when they will cause disruption of the positive feedback effects of estradiol, thus blocking ovulation in adult life. The resulting ovarian morphology thus appears to be like that of PCOS but is in fact not a clear mimic. This review of the various studies highlights parallels and problems with the use of rodents to study the mechanisms underlying the development of PCOS in women.

Effect of aging on follicular function may be relieved by exogenous gonadotropin treatment in a sheep model.

The current study investigated hormonal and ovarian changes during physiological reproductive aging in Sarda ewes. In a first experiment, follicular and corpus luteum dynamics were compared during an induced oestrus cycle in aged (12-14 years) and young adult ewes (4-5 years). Oestrus cycle characteristics did not differ between the two experimental groups. However, follicular function during the follicular phase showed significant alterations in aged ewes, as determined by a lack of dominance effect and by lower mean values of circulating oestradiol (E(2)) and inhibin levels, compared with young adult ewes. In a second experiment, differences in follicle growth, hormonal milieu and oocyte quality in response to exogenous FSH administration were assessed in aged and adult ewes. No differences were recorded in ovarian response to FSH treatment between young adult and aged ewes, as evaluated by ultrasonographic data and circulating concentrations of LH, E(2) and inhibin-A. Although the total number of recovered oocytes was similar in the two age groups, the number of good quality oocytes selected for IVM was significantly lower in aged ewes compared with adult ones. Thereafter, no differences were recorded in cleavage rates, total blastocyst output, embryo developmental kinetic and quality between aged and adult groups. In conclusion, this study demonstrated that reproductive aging in sheep is associated with impaired follicle functionality and an increase in the proportion of oocytes showing morphological abnormalities. However interestingly, oocyte developmental competence in vitro and embryo cryotolerance were not affected by the aging process, when only good quality oocytes were chosen.

Expression of guanylyl cyclase-B (GC-B/NPR2) receptors in normal human fetal pituitaries and human pituitary adenomas implicates a role for C-type natriuretic peptide.

C-type natriuretic peptide (CNP/Nppc) is expressed at high levels in the anterior pituitary of rats and mice and activates guanylyl cyclase B receptors (GC-B/Npr2) to regulate hormone secretion. Mutations in NPR2/Npr2 can cause achondroplasia, GH deficiency, and female infertility, yet the normal expression profile within the anterior pituitary remains to be established in humans. The current study examined the expression profile and transcriptional regulation of NPR2 and GC-B protein in normal human fetal pituitaries, normal adult pituitaries, and human pituitary adenomas using RT-PCR and immunohistochemistry. Transcriptional regulation of human NPR2 promoter constructs was characterized in anterior pituitary cell lines of gonadotroph, somatolactotroph, and corticotroph origin. NPR2 was detected in all human fetal and adult pituitary samples regardless of age or sex, as well as in all adenoma samples examined regardless of tumor origin. GC-B immunoreactivity was variable in normal pituitary, gonadotrophinomas, and somatotrophinomas. Maximal transcriptional regulation of the NPR2 promoter mapped to a region within -214 bp upstream of the start site in all anterior pituitary cell lines examined. Electrophoretic mobility shift assays revealed that this region contains Sp1/Sp3 response elements. These data are the first to show NPR2 expression in normal human fetal and adult pituitaries and adenomatous pituitary tissue and suggest a role for these receptors in both pituitary development and oncogenesis, introducing a new target to manipulate these processes in pituitary adenomas.

Inactivation of AMPKα1 induces asthenozoospermia and alters spermatozoa morphology.

AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis, is present in metabolic tissues (muscle and liver) and has been identified as a modulator of the female reproductive functions. However, its function in the testis has not yet been clearly defined. We have investigated the potential role of AMPK in male reproduction by using transgenic mice lacking the activity of AMPK catalytic subunit α1 gene [α1AMPK knockout (KO)]. In the testis, the α1AMPK subunit is expressed in germ cells and also in somatic cells (Sertoli and Leydig cells). α1AMPK KO male mice show a decrease in fertility, despite no clear alteration in the testis morphology or sperm production. However, in α1AMPK(-/-) mice, we demonstrate that spermatozoa have structural abnormalities and are less motile than in control mice. These spermatozoa alterations are associated with a 50% decrease in mitochondrial activity, a 60% decrease in basal oxygen consumption, and morphological defects. The α1AMPK KO male mice had high androgen levels associated with a 5- and 3-fold increase in intratesticular cholesterol and testosterone concentrations, respectively. High concentrations of proteins involved in steroid production (3ß-hydroxysteroid dehydrogenase, cytochrome steroid 17 alpha-hydroxylase/17,20 lysate, and steroidogenic acute regulatory protein) were also detected in α1AMPK(-/-) testes. In the pituitary, the LH and FSH concentrations tended to be lower in α1AMPK(-/-) male mice, probably due to the negative feedback of the high testosterone levels. These results suggest that total α1AMPK deficiency in male mice affects androgen production and quality of spermatozoa, leading to a decrease in fertility.

Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

Prolactin (PRL) is mainly expressed in the pituitary in rodents, whereas in humans, expression is observed in many extrapituitary sites, including lymphocytes. Due to the lack of adequate experimental models, the function of locally produced PRL in the immune system is largely unknown. Using transgenic rats that express luciferase under the control of extensive human PRL regulatory regions, we characterized immune cell responses to thioglycollate (TG)-induced peritonitis. Resident populations of myeloid cells in the peritoneal cavity of untreated rats expressed barely detectable levels of luciferase. In contrast, during TG-induced peritonitis, cell-specific expression in both neutrophils and monocytes/macrophages in peritoneal exudates increased dramatically. Elevated luciferase expression was also detectable in peripheral blood and bone marrow CD11b(+) cells. Ex vivo stimulation of primary myeloid cells showed activation of the human extrapituitary promoter by TNF-α, lipopolysaccharide, or TG. These findings were confirmed in human peripheral blood monocytes, showing that the transgenic rat provided a faithful model for the human gene. Thus, the resolution of an inflammatory response is associated with dramatic activation of the PRL gene promoter in the myeloid lineage.

Diagnostic applications for inhibin and activins.

Inhibin and activins play major roles as paracrine and autocrine signaling molecules in reproduction and development where the main emphasis has been placed in developing potential diagnostic applications. While a role for activin assays in diagnostics has so far been unfounded, ELISAs specific for the biologically active inhibin A and B dimers, and for free inhibin alpha subunits, alone or in combination have found some specific diagnostic applications. Addition of inhibin A to the triple test for Down syndrome in the second trimester of pregnancy, measurement of total inhibin as a marker of certain forms of ovarian cancer in specific circumstances, and inhibin B for male fertility are useful diagnostics. A review of the evidence so far suggests that other applications for inhibin and activin assays have yet to be confirmed, or translated into reliable tools for clinical practice.

The fate of granulosa cells following premature oocyte loss and the development of ovarian cancers.

This review examines the importance of the epithelial origin of granulosa cells and their possible contribution to the development of ovarian cancers in three animal models. We hypothesise that undifferentiated granulosa cells, devoid of their germ cell regulator, retain their embryonic plasticity and may give rise to ovarian cancers of epithelial origin. Dazl-KO and FancD2-KO mice and BMP15-KO sheep are animal models in which germ cells or oocytes are lost at specific stages of follicular formation or growth, leaving behind clusters of residual, but healthy somatic cells. A common feature in Dazl- and Fancd2-KO animals following germ cell/oocyte loss is the presence of sex cords and intraovarian epithelial ducts or tubules. In Dazl-KO mice, cord/tubule-like structures, OSE invaginations and clusters of steroidogenic cells became increasingly prominent with age, but these abnormal structures remained benign. In Fancd2-KO mice, the formation of sex-cords and intraovarian tubules lead to the formation of tumours with multiple phenotypes including luteomas, papillary cysts and malignant carcinomas (e.g. adenocarcinomas). In BMP15-KO sheep, oocytes die as follicles start to grow, leaving 'nodules' containing granulosa cells with a capacity to respond to follicle stimulating hormone and synthesize inhibin. Thereafter, these nodules coalesced and a range of benign solid or semi-solid tumour phenotypes developed. We conclude that premature loss of oocytes, but not granulosa cells, leads to tumour formation with multiple phenotypes. Moreover, the severity of tumour development is linked to both the specificity of the mutation and the timing of oocyte loss relative to that of follicular formation.

Enhanced thecal androgen production is prenatally programmed in an ovine model of polycystic ovary syndrome.

One of the hallmarks of polycystic ovary syndrome (PCOS) is increased ovarian androgen secretion that contributes to the ovarian, hormonal, and metabolic features of this condition. Thecal cells from women with PCOS have an enhanced capacity for androgen synthesis. To investigate whether this propensity is a potential cause, rather than a consequence, of PCOS, we used an ovine prenatal androgenization model of PCOS and assessed ewes at 11 months of age. Pregnant Scottish Greyface ewes were administered 100 mg testosterone propionate (TP) or vehicle control twice weekly from d 62 to 102 of gestation, and female offspring (TP = 9, control = 5) were studied. Prenatal TP exposure did not alter ovarian morphology or cyclicity, or plasma androgen, estrogen, and gonadotropin concentrations, at this stage. However, follicle function was reprogrammed in vivo with increased proportions of estrogenic follicles (P < 0.05) in the TP-exposed cohort. Furthermore, in vitro the thecal cells of follicles (>4 mm) secreted more LH-stimulated androstenedione after prenatal androgenization (P < 0.05), associated with increased basal expression of thecal StAR (P < 0.01), CYP11A (P < 0.05), HSD3B1 (P < 0.01), CYP17 (P < 0.05), and LHR (P < 0.05). This provides the first evidence of increased thecal androgenic capacity in the absence of a PCOS phenotype, suggesting a thecal defect induced during fetal life.

Effect of androgen treatment during foetal and/or neonatal life on ovarian function in prepubertal and adult rats.

We investigated the effects of different windows of testosterone propionate (TP) treatment during foetal and neonatal life in female rats to determine whether and when excess androgen exposure would cause disruption of adult reproductive function. Animals were killed prepubertally at d25 and as adults at d90. Plasma samples were taken for hormone analysis and ovaries serial sectioned for morphometric analyses. In prepubertal animals, only foetal+postnatal and late postnatal TP resulted in increased body weights, and an increase in transitory, but reduced antral follicle numbers without affecting total follicle populations. Treatment with TP during both foetal+postnatal life resulted in the development of streak ovaries with activated follicles containing oocytes that only progressed to a small antral (smA) stage and inactive uteri. TP exposure during foetal or late postnatal life had no effect upon adult reproductive function or the total follicle population, although there was a reduction in the primordial follicle pool. In contrast, TP treatment during full postnatal life (d1-25) resulted in anovulation in adults (d90). These animals were heavier, had a greater ovarian stromal compartment, no differences in follicle thecal cell area, but reduced numbers of anti-Mullerian hormone-positive smA follicles when compared with controls. Significantly reduced uterine weights lead reduced follicle oestradiol production. These results support the concept that androgen programming of adult female reproductive function occurs only during specific time windows in foetal and neonatal life with implications for the development of polycystic ovary syndrome in women.