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

A screen for round egg mutants in Drosophila identifies tricornered, furry, and misshapen as regulators of egg chamber elongation.

The elongation of tissues and organs during embryonic development results from the coordinate polarization of cell behaviors with respect to the elongation axis. Within the Drosophila melanogaster ovary, initially spherical egg chambers lengthen dramatically as they develop to create the elliptical shape of the mature egg. This morphogenesis depends on an unusual form of planar polarity within the egg chamber's outer epithelial cell layer known as the follicle cells. Disruption of follicle cell planar polarity leads to the production of round rather than elongated eggs; however, the molecular mechanisms that control this tissue organization are poorly understood. Starting from a broadly based forward genetic screen, we have isolated 12 new round egg complementation groups, and have identified four of the mutated genes. In mapping the largest complementation group to the fat2 locus, we unexpectedly discovered a high incidence of cryptic fat2 mutations in the backgrounds of publicly available stocks. Three other complementation groups correspond to the genes encoding the cytoplasmic signaling proteins Tricornered (Trc), Furry (Fry), and Misshapen (Msn). Trc and Fry are known members of an NDR kinase signaling pathway, and as a Ste20-like kinase, Msn may function upstream of Trc. We show that all three proteins are required for follicle cell planar polarity at early stages of egg chamber elongation and that Trc shows a planar polarized distribution at the basal follicle cell surface. These results indicate that this new mutant collection is likely to provide novel insight into the molecular mechanisms controlling follicle cell planar polarity and egg chamber elongation

Improving the reproductive performance of the Muturu breed of cattle in Nigeria using modified Ovsynch and progesterone synchronization protocol : a thesis presented in partial fulfilment of the requirement for the degree of Master of Science in Animal Science, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand

The Ovsynch protocol has been used in cattle to synchronize ovulation and facilitate fixed-time AI (FTAI) but its efficacy has not been evaluated in the Nigerian Muturu breed of B. indicus cattle. The effects of a modified Ovsynch protocol (modified by the addition of progesterone, eCG and modification of time for AI) upon the reproductive performance of Muturu cows were therefore studied. The study was conducted at Abakiliki in Ebonyi state, Nigeria. Muturu cows (n=100) were selected for the study based on their previous calving history and the presence of follicle of greater than 5 mm diameter. Cows were then allotted to Untreated and Treated groups (n=50 each). Synchronization was by an Ovsynch regimen (Day 0: 100 μg GnRH, Day 7: 500 μg PGF2α (Ovuprost), Day 10: 100 μg GnRH), augmented by a progesterone-releasing intravaginal insert (CIDR) between Days 0 and 7, and 400 IU eCG (Norvomon) on Day 7. FTAI was performed 12 and 24 h after the second GnRH injection. Untreated animals were monitored over two consecutive oestrous cycles and examined daily for the presence of oestrus over a period of 49 days, and were exposed to natural mating upon detection of oestrus in the second oestrous cycle observed. Ovarian ultrasonography to ascertain follicle size was performed at the onset and end of oestrus in the Untreated group, and on Days 0,7,10 in the Treated group. Pregnancy diagnosis (ultrasonographic) was performed 45 days after FTAI (Synchronized) and 45 days after the last observed oestrus during the breeding season (Untreated). Blood samples were collected from Treated group of animals for progesterone, LH and oestradiol concentrations assay. All animals in the Treated group displayed oestrus after synchronization and all animals in the Untreated groups displayed oestrus during the 49-day study period. Follicle size (18 ± 0.4 mm versus 12± 0.2 mm), ovulation rate (100% versus 64%), duration of oestrus (54 h versus 19 h ) and 45-day pregnancy rate (84% versus 36%), were all greater (p<0.005) in Treated than Untreated animals. Additionally, the animals in the Treated group displayed 46% multiple ovulations, compared with none in the Untreated group. Oestradiol concentrations were related to follicle size. Increase in follicle size resulted in higher concentration of oestradiol. The presence of multiple ovulations appears to have been related to the use of eCG in the protocol. No multiple pregnancies occurred. It is concluded that the modified Ovsynch protocol produced synchrony of oestrus, ovulation and improved follicle size, that were supported by normal endocrine patterns. It therefore appears that oestrus synchronization and FTAI can improve reproductive performance of the Muturu breed of cattle. Key words: Muturu cows, pregnancy, follicle size, ovulation, oestrus, modified Ovsynch, Nigeri

Computer-Generated Ovaries to Assist Follicle Counting Experiments

Precise estimation of the number of follicles in ovaries is of key importance in the field of reproductive biology, both from a developmental point of view, where follicle numbers are determined at specific time points, as well as from a therapeutic perspective, determining the adverse effects of environmental toxins and cancer chemotherapeutics on the reproductive system. The two main factors affecting follicle number estimates are the sampling method and the variation in follicle numbers within animals of the same strain, due to biological variability. This study aims at assessing the effect of these two factors, when estimating ovarian follicle numbers of neonatal mice. We developed computer algorithms, which generate models of neonatal mouse ovaries (simulated ovaries), with characteristics derived from experimental measurements already available in the published literature. The simulated ovaries are used to reproduce in-silico counting experiments based on unbiased stereological techniques; the proposed approach provides the necessary number of ovaries and sampling frequency to be used in the experiments given a specific biological variability and a desirable degree of accuracy. The simulated ovary is a novel, versatile tool which can be used in the planning phase of experiments to estimate the expected number of animals and workload, ensuring appropriate statistical power of the resulting measurements. Moreover, the idea of the simulated ovary can be applied to other organs made up of large numbers of individual functional units

RFRP3 influences basal lamina degradation, cellular death, and progesterone secretion in cultured preantral ovarian follicles from the domestic cat.

The hypothalamic neuropeptide RFRP3 can suppress hypothalamic GnRH neuron activation and inhibit gonadotropin release from the anterior pituitary. RFRP3 is also produced locally in the ovary and can inhibit steroidogenesis and follicle development in many vertebrates. However, almost nothing is known about the presence and regulatory action of RFRP3 in gonads of any carnivore species. Such knowledge is important for developing captive breeding programs for endangered carnivores and for inhibiting reproduction in feral species. Using the domestic cat as a model, our objectives were to (1) demonstrate the expression of feline RFRP3 (fRFRP3) and its receptor in the cat ovary and (2) assess the influence of fRFRP3 on ovarian follicle integrity, survival, and steroidogenesis in vitro. We first confirmed that fRFRP3 and its receptors (NPFFR1 and NPFFR2) were expressed in cat ovaries by sequencing PCR products from ovarian RNA. We then isolated and cultured preantral ovarian follicles in the presence of 10 or 1 µM fRFRP3 + FSH (1 µg/mL). We recorded the percentage of morphologically viable follicles (basal lamina integrity) over 8 days and calculated percentage survival of follicles on Day 8 (using fluorescent markers for cell survival and death). Last, we quantified progesterone accumulation in media. 10 µM fRFRP3 had no observable effect on viability, survival, or steroid production compared to follicles exposed to only FSH. However, 1 µM fRFRP3 decreased the percentage of morphologically viable follicles and the percentage of surviving follicles on Day 8. At the same time, 1 µM fRFRP3 increased the accumulation of progesterone in media. Our study shows, for the first time, direct action of RFRP3 on the follicle as a functional unit, and it is the first in a carnivore species. More broadly, our results support a conserved, inhibitory action of RFRP3 on ovarian follicle development and underscore the importance of comparative functional studies

Perturbations in Lineage Specification of Granulosa and Theca Cells May Alter Corpus Luteum Formation and Function

Anovulation is a major cause of infertility, and it is the major leading reproductive disorder in mammalian females. Without ovulation, an oocyte is not released from the ovarian follicle to be fertilized and a corpus luteum is not formed. The corpus luteum formed from the luteinized somatic follicular cells following ovulation, vasculature cells, and immune cells is critical for progesterone production and maintenance of pregnancy. Follicular theca cells differentiate into small luteal cells (SLCs) that produce progesterone in response to luteinizing hormone (LH), and granulosa cells luteinize to become large luteal cells (LLCs) that have a high rate of basal production of progesterone. The formation and function of the corpus luteum rely on the appropriate proliferation and differentiation of both granulosa and theca cells. If any aspect of granulosa or theca cell luteinization is perturbed, then the resulting luteal cell populations (SLC, LLC, vascular, and immune cells) may be reduced and compromise progesterone production. Thus, many factors that affect the differentiation/lineage of the somatic cells and their gene expression profiles can alter the ability of a corpus luteum to produce the progesterone critical for pregnancy. Our laboratory has identified genes that are enriched in somatic follicular cells and luteal cells through gene expression microarray. This work was the first to compare the gene expression profiles of the four somatic cell types involved in the follicle-to-luteal transition and to support previous immunofluorescence data indicating theca cells differentiate into SLCs while granulosa cells become LLCs. Using these data and incorporating knowledge about the ways in which luteinization can go awry, we can extrapolate the impact that alterations in the theca and granulosa cell gene expression profiles and lineages could have on the formation and function of the corpus luteum. While interactions with other cell types such as vascular and immune cells are critical for appropriate corpus luteum function, we are restricting this review to focus on granulosa, theca, and luteal cells and how perturbations such as androgen excess and inflammation may affect their function and fertility

The value of automated follicle volume measurements in IVF/ICSI

Background/Aims: The objective of this literature study is to investigate the place of recent software technology sonography-based automated volume count (SonoAVC) for the automatic measurement of follicular volumes in IVF/ICSI. Its advantages and disadvantages and potential future developments are evaluated. Methods: A total of 74 articles were read via a PubMed literature study.The literature study included 53 articles, 32 of which for the systematic review. Results: The SonoAVC software shows excellent accuracy. Comparing the technology with the “golden standard” two-dimensional (2D) manual follicle measurements, SonoAVC leads to a significantly lower intra- and inter-observer variability. However, there is no significant difference in clinical outcome (pregnancy rate).We noted a significant advantage in the time gained, both for doctor and patient. By storing the images, the technology offers the possibility of including a quality control and continuous training and further standardization of follicular monitoring can be expected. Ovarian reserve testing by measuring the antral follicle count with SonoAVC is highly reliable. Conclusion: This overview of previously published literature shows how SonoAVC offers advantages for clinical practice, without losing any accuracy or reliability. Doctors should be motivated to the general use of follicular volumes instead of follicular diameters

Primordial follicular assembly in humans : revisited

Peer reviewedPreprin

Transient activaton of β-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours

When β-catenin signalling is disturbed from mid-gestation onwards lineage commitment is profoundly altered in postnatal mouse epidermis. We have investigated whether adult epidermis has the capacity for β-catenin-induced lineage conversion without prior embryonic priming. We fused N-terminally truncated, stabilised β-catenin to the ligand-binding domain of a mutant oestrogen receptor (ΔNβ -cateninER). ΔNβ-cateninER was expressed in the epidermis of transgenic mice under the control of the keratin 14 promoter and β -catenin activity was induced in adult epidermis by topical application of 4-hydroxytamoxifen (4OHT). Within 7 days of daily 4OHT treatment resting hair follicles were recruited into the hair growth cycle and epithelial outgrowths formed from existing hair follicles and from interfollicular epidermis. The outgrowths expressed Sonic hedgehog, Patched and markers of hair follicle differentiation, indicative of de novo follicle formation. The interfollicular epidermal differentiation program was largely unaffected but after an initial wave of sebaceous gland duplication sebocyte differentiation was inhibited. A single application of 4OHT was as effective as repeated doses in inducing new follicles and growth of existing follicles. Treatment of epidermis with 4OHT for 21 days resulted in conversion of hair follicles to benign tumours resembling trichofolliculomas. The tumours were dependent on continuous activation of β-catenin and by 28 days after removal of the drug they had largely regressed. We conclude that interfollicular epidermis and sebaceous glands retain the ability to be reprogrammed in adult life and that continuous β-catenin signalling is required to maintain hair follicle tumours.Peer reviewedFinal Published versio

Species-specific differences in follicular antral sizes result from diffusion-based limitations on the thickness of the granulosa cell layer

The size of mature oocytes is similar across mammalian species, yet the size of ovarian follicles increases with species size, with some ovarian follicles reaching diameters more than 1000-fold the size of the enclosed oocyte. Here we show that the different follicular sizes can be explained with diffusion-based limitations on the thickness of the hormone-secreting granulosa layer. By analysing published data on human follicular growth and granulosa cell expansion during follicular maturation we find that the 4-fold increase of the antral follicle diameter is entirely driven by an increase in the follicular fluid volume, while the thickness of the surrounding granulosa layer remains constant at about 45+/-10 mkm. Based on the measured kinetic constants, the model reveals that the observed fall in the gonadotropin concentration from peripheral blood circulation to the follicular antrum is a result of sequestration in the granulosa. The model further shows that as a result of sequestration, an increased granulosa thickness cannot substantially increase estradiol production but rather deprives the oocyte from gonadotropins. Larger animals (with a larger blood volume) require more estradiol as produced by the ovaries to downregulate FSH-secretion in the pituitary. Larger follicle diameters result in larger follicle surface areas for constant granulosa layer thickness. The reported increase in follicular surface area in larger species indeed correlates linearly both with species mass and with the predicted increase in estradiol output. In summary, we propose a structural role for the antrum in that it determines the volume of the granulosa layer and thus the level of estrogen production.Comment: Mol Hum Repr 201