XIV Workshop on the Development and Function of Reproductive Organs

During all periods of male ontogenesis, physiological processes responsible for the correct functioning of reproductive organs and spermatogenesis are under the influence of various factors (neuro-humoral, genetic, and paratypical). Recently, the attention of researchers has increasingly turned to the study of epigenetic factors. In scientific publications, one can increasingly find references to the direct role of microRNAs, small non-coding RNAs involved in post-transcriptional regulation of gene expression, in the processes of development and functioning of reproductive organs. Although the role of microRNAs in the reproduction of mammals, including humans, has been intensively studied, this area of knowledge in birds remains under-researched and limited to single experiments. This is likely due to the unique features of embryogenesis and the structure of the avian reproductive system. This review summarizes the current state of knowledge on the role of microRNAs in avian reproduction. Insight into the molecular basis of spermatogenesis in Gallus gallus domesticus is provided. Data on the functions and mechanisms by which microRNAs influence the processes of growth, development, and formation of rooster germ cells that determine the necessary morphofunctional qualitative characteristics of mature spermatozoa are summarized. Particular attention is paid to miRNA biogenesis as an important step affecting the success of spermatogenesis, as well as the role of miRNAs in avian sex differentiation during early embryogenesis. The modern literature sources systematized in this review, revealing the questions about the role of miRNAs in the reproductive function of birds, create a theoretical basis and define new perspectives and directions for further research in this field.

Germline development from human pluripotent stem cells toward disease modeling of infertility

Primordial germ cells (PGCs) are the embryonic progenitors of sperm and egg cells. Mammalian PGCs are thought to actively migrate from the yolk sac endoderm over long distances across the embryo to reach the somatic genital ridges. The general principles of mammalian PGC development were discovered in mice. In contrast, little is known about PGC development in primates due to extremely limited access to primate embryos. Here, we analyzed 12 well preserved marmoset monkey (Callithrix jacchus) embryos covering the phase from PGC emergence in the endoderm to the formation of the sexually differentiated gonad (embryonic day (E) 50 to E95). We show using immunohistochemistry that the pluripotency factors OCT4A and NANOG specifically mark PGCs throughout the period studied. In contrast, SALL4 and LIN28 were first expressed ubiquitously and only later down-regulated in somatic tissues. We further show, for the first time, that PGCs are located in the endoderm in E50 embryos in close spatial proximity to the prospective genital ridge, making a long-range migration of PGCs dispensable. At E65, PGCs are already present in the primitive gonad, while significantly later embryonic stages still exhibit PGCs at their original endodermal site, revealing a wide spatio-temporal window of PGC distribution. Our findings challenge the ‘dogma’ of active long-range PGC migration from the endoderm to the gonads. We therefore favor an alternative model based primarily on passive translocation of PGCs from the mesenchyme that surrounds the gut to the prospective gonad through the intercalar expansion of mesenchymal tissue which contains the PGCs. In summary, we (i) show differential pluripotency factor expression during primate embryo development and (ii) provide a schematic model for embryonic PGC translocation.

Primordial germ cells (PGCs) are the embryonic progenitors of sperm and egg cells. Mammalian PGCs are thought to actively migrate from the yolk sac endoderm over long distances across the embryo to reach the somatic genital ridges. The general principles of mammalian PGC development were discovered in mice. In contrast, little is known about PGC development in primates due to extremely limited access to primate embryos. Here, we analyzed 12 well preserved marmoset monkey (Callithrix jacchus) embryos covering the phase from PGC emergence in the endoderm to the formation of the sexually differentiated gonad (embryonic day (E) 50 to E95). We show using immunohistochemistry that the pluripotency factors OCT4A and NANOG specifically mark PGCs throughout the period studied. In contrast, SALL4 and LIN28 were first expressed ubiquitously and only later down-regulated in somatic tissues. We further show, for the first time, that PGCs are located in the endoderm in E50 embryos in close spatial proximity to the prospective genital ridge, making a long-range migration of PGCs dispensable. At E65, PGCs are already present in the primitive gonad, while significantly later embryonic stages still exhibit PGCs at their original endodermal site, revealing a wide spatio-temporal window of PGC distribution. Our findings challenge the ‘dogma’ of active long-range PGC migration from the endoderm to the gonads. We therefore favor an alternative model based primarily on passive translocation of PGCs from the mesenchyme that surrounds the gut to the prospective gonad through the intercalar expansion of mesenchymal tissue which contains the PGCs. In summary, we (i) show differential pluripotency factor expression during primate embryo development and (ii) provide a schematic model for embryonic PGC translocation.

Many attempts to establish embryonic stem (ES) cells from pre-implantation stage embryos in pigs have failed. An alternate source of pluripotent stem cells is embryonic germ (EG) cells derived from primordial germ cells (PGCs) of the genital ridge, which is developed from the mesonephros. Mesonephros is a vestigial, transient renal organ that functions only during embryonic development. It is believed to be a source of multiple stem cells including somatic cells in the gonad, vascular endothelial cells, and hematopoietic stem cells. Therefore, we tried to obtain putative stem cells from cells isolated from porcine mesonephros under culture conditions used to establish EG cells. Porcine fetuses from crossbred gilts were collected by hysterectomy between Days 25 and 30 of pregnancy (estrus = Day 0). Mesonephros and genital ridges were separated from each other, and cells from the mesonephros and PGCs from genital ridges were isolated by a physical method. Isolated cells were cultured in PES medium [50:50 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F10 medium supplemented with 15% fetal bovine serum (FBS), l-glutamine (1.7 mM), β-mercaptoethanol (0.1 mM), 1% MEM non-essential amino acids and 1% antibiotic-antimycotic] containing the cytokines, soluble recombinant human basic fibroblast growth factor (bFGF; 20 ng/mL), and human leukemia inhibitory factor (hLIF; 10 ng/mL). Isolated cells were cultured on fresh primary murine embryonic fibroblast feeder cells (PMEF) in a humidified environment of 5% CO2 in air at 38°C. The colonies with EG-like morphology were dissociated with 0.25% trypsin/1 mM EDTA for 10 min, and passed to fresh feeders. After 5–8 days, colonies started to grow with typical EG-like morphology. More colonies were obtained from the culture of cells from mesonephros than from culture of PGCs. Porcine EG-like cells from mesonephros (pMN-EG-like cells) were passed to fresh feeder every 6–8 days and have been cultured up to 9 passages while maintaining typical EG-like morphology. pMN-EG-like cells were stained for alkaline phosphatase throughout the culture. Furthermore, these cells reacted with antibodies against Oct-4 and SSEA-1 by immunocytochemistry, indicating that these cells have characteristics of pluripotential stem cells. In order to characterize the pMN-EG-like cells with respect to their potential for differentiation, embryoid body (EB) formation was induced. EBs started to form in 4 days and cystic structures in 2 weeks. EBs were then attached to the dish and cultured without cytokines. Spontaneously, EBs from pMN-EG-like cells could give rise to differentiated cell types such as neuronal-like, epithelial-like, and fibroblast-like cells. Further studies to characterize differentiated cells from pMN-EG-like cells by immunocytochemistry and for teratoma formation by injection into SCID mice will be performed. In conclusion, EG-like cells could be obtained from culture of mesonephric cells from porcine fetus and further characterization of these cells is required.

The insect growth regulator NC-184, a juvenile hormone mimic, prevents moulting to the adult stage in the desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae). Male nymphs treated in the penultimate or final nymphal instar with NC-184 exhibit precocious mating behaviour in the final instar. We examined whether this chemical affects the development of the internal reproductive organs of crowded nymphs. In treated males, both accessory glands and seminal vesicles were underdeveloped, and no sperm was found in the seminal vesicle, whereas these organs in control individuals had greatly increased in size 10 days after treatment, when all the insects had moulted to adults. Testis size in treated males was similar to that in controls, regardless of their smaller body size due to the inhibition of moulting. Oogenesis and development of spermatheca in females treated with NC-184 continued to some degree, but no eggs matured, unlike what occurred in the control. In conclusion, treatment of S. gregaria nymphs with NC-184 resulted in changes in the reproductive organs in both sexes.

Primordial germ cells (PGCs) are useful for producing transgenic chickens and preserving genetic material in avian species. In this study, we investigated the in vitro differentiation potential of chicken PGCs into different cell types. For differentiation into adipocytes, chicken PGCs were cultured for 21 days in induction media containing dexamethasone, insulin and/or 3-isobutyl-1-methylxanthine (IBMX), and differentiation rates ranging from 74% to 91% were identified by oil red-O and alkaline phosphatase (ALP) staining. For differentiation into neuron-like cells, chicken PGCs were cultured for 3 or 7 days in the induction media containing retinoic acid (RA) and IBMX, and differentiation rates ranging from 71% to 87% were identified by toluidine blue staining and immunohistochemical staining. For differentiation into osteoblasts, chicken PGCs were cultured for 15 or 21 days in the induction media containing desamethasone, beta-glycerol phosphate and/or vitamin C, and differentiation rates ranging from 47% to 79% were confirmed by Von Kossa, cytochemical and immunohistochemical staining. These data suggest that, like mammalian PGCs, chicken PGCs can differentiate into different cell types in vitro.

T-2 toxin, an inevitable environmental pollutant, is the most toxic type A trichothecene mycotoxin. Reproductive disruption is a key adverse effect of T-2 toxin. Herein, this paper reviews the reproductive toxicity of T-2 toxin and its mechanisms in male and female members of different species. The reproductive toxicity of T-2 toxin is evidenced by decreased fertility, disrupted structures and functions of reproductive organs, and loss of gametogenesis in males and females. T-2 toxin disrupts the reproductive endocrine axis and inhibits reproductive hormone synthesis. Furthermore, exposure to T-2 toxin during pregnancy results in embryotoxicity and the abnormal development of offspring. We also summarize the research progress in counteracting the reproductive toxicity of T-2 toxin. This review provides information toward a comprehensive understanding of the reproductive toxicity mechanisms of T-2 toxin.

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

β-importin Tnpo-SR promotes germline stem cell maintenance and oocyte differentiation in female Drosophila

Primordial germ cells represent the founder population for establishing the germ line providing the continuity of life between generations. PG2, a germ cell-specific antigen, is one of the few continuously detectable epitopes in mammalian primordial germ cells and it is dynamically expressed during early post-fertilization development and during postnatal germ cell maturation. Immunoelectron microscopy shows a localization of PG2 in the peri-mitochondrial cytoplasm but its further subcellular or biochemical nature remains elusive. For further characterization of the PG2 epitope we used regular and semi-thin cryosection of ovulated and isolated follicular rabbit oocytes and localized all mitochondria with the help of the constitutive mitochondrial antigen MTC02 in double immunofluorescence stainings. Semi-thin cryosections of ovulated oocytes revealed a general close co-localization of both antibody reactions at the level of single mitochondria. In centrifuged follicular oocytes both antigens co-sedimented almost completely indicating a topographical association of the epitopes on the basis of a strong interaction of PG2 with mitochondria. To begin to characterize the germ cell epitope biochemically we treated oocyte cryosections either with acetone to reduce lipids or with N-glycosidase F to remove N-linked glycosylations before the immuoreaction. Neither treatment affected the antibody characteristics, which suggests that the PG2 epitope is most probably a protein. Because of the close interaction of PG2 with the mitochondria we speculate that PG2 is involved in the change of the mitochondrial morphology typically observed during differentiation of germ cells.

![][1] The Spanish National Cancer Institute (CNIO) Meeting on Development and Cancer took place between 4 and 6 February 2008 in Madrid, Spain, and was organized by K. Basler, G. Morata, E. Moreno and M. Torres. ![][2] Developmental biologists rightly argue that genes regulating developmental pathways are reiterated in cancer development. At the recent Spanish National Cancer Institute (CNIO) Meeting on Development and Cancer, novel concepts and processes that are considered to be common to both embryonic and cancer development were discussed. It is worth pointing out that tumour formation should be considered to be a form of aberrant organ development, in which many developmental pathways that control proliferation, differentiation and growth or morphogenesis are affected. This report is structured in three parts, describing the work presented at the meeting according to the stages of tumour development to which it pertains. First, the normal behaviour of cell populations that are the targets of transformation, in particular adult stem cells and the pathways that regulate their behaviour. Second, processes that occur at the early stages of cancer development before morphological malformations are detectable, in particular the process that is best known from studies in flies as ‘cell competition’. Third, developmental processes and pathways that are active in the growing tumour—which we consider to be an extrinsic organ—for example, the recruitment of blood vessels or the role of morphogens in controlling the size and shape of tumours, and the progression from hyperplasia to invasive tumours (Fig 1). Figure 1. Tumour development and genes involved in the different stages. Schematic representation of how stem cells (A) , by acquiring a mutation through cell competition ( B ) and by the selection of additional mutations, can give rise to the ‘tumour organ’ ( C ). The genes involved in these processes are listed on the right‐hand side, and most of them … [1]: /embed/graphic-1.gif [2]: /embed/graphic-2.gif

Improving gilt development is key to maximizing sow longevity. How to best manage and feed gilts during development is heavily debated. A series of studies were conducted to determine the optimal feeding program for gilts during development. The hypothesis was that metabolizable energy (ME) and standard ileal digestible lysine could be varied under commercial conditions (ad libitum feeding) to optimize body weight gain and composition, age at puberty, and mammary gland development to support increased productivity of gilts. Studies were conducted in commercial production facilities in Iowa and Utah using large numbers of maternal-line gilts that were monitored, under various dietary treatments from 100 to 200 d of age, for growth, body composition, age at puberty and development of reproductive organs and mammary glands. Varying the dietary Lysine:ME ratio (g:mcal) had modest effects on growth and body composition of gilts. Likewise, development of reproductive organs was minimally affected by diet, but slower growth delayed development of terminal lobular ductal units in mammary glands. Growth rates were slowed by reducing dietary lysine:ME ratio below the requirement and increasing fiber. Gilts still had adequate growth (> 600 g/d) and body composition (≥ 15 mm backfat) for good reproductive performance. Although dietary treatments did not affect the average age of puberty (205 d) or the total number of gilts that reached puberty, a greater percentage of gilts fed higher lysine diets exhibited puberty during the first 40 days of boar exposure. Gilts lost 2 to 3 mm backfat once moved from the development unit to the breeding farms, but pregnancy rates were ≥ 95%. Culling of gilts before first breeding did not vary with diet but was considerably higher than expected. Investigation of how these developmental traits affect productivity through subsequent parities is ongoing.

Introduction: Repeated heat shock (HS) stresses reduce the reproduction rate of Drosophila flies. Heat shock proteins (HSPs) protect cells against irreversible damages inducing heatinduced.Oxidative stress declines protective function of HSPs. Diploschistes ocellatus lichen aqueous extract possesses a strong antioxidant potential in vitro. Antioxidants can preserve HSPs function. Therefore, the present study for the first time investigated the cytoprotective effects of D. ocellatus aqueous extract against HS-mediated deleterious effects on reproductive function in Drosophila melanogaster. Methods: Three different types of culture media including control, 30% lichen extract, and 60%lichen extract were prepared. Adult D. melanogaster flies were placed on Delcour medium and allowed to lay eggs for 2 hours. Then the eggs were equally distributed between the culture media. After flies completed their life cycle, the adult enclosed flies were exposed to HS. To assess reproductive function, the newly emerged adult flies were transferred to the freshly prepared regular culture medium every three days for 3 times and finally adult offspring born to these flies were enumerated.Results: HS negatively affected the reproduction rate in flies in control group. Quantification of adult enclosed flies born to the D. ocellatus extract treated flies showed that lichen extract could negate the deleterious effects of HS on reproduction function of D. melanogaster in a dose-dependent manner.Conclusion: Diploschistes ocellatus aqueous extract attenuated the harmful effects of HS stress on reproductive function of D. melanogaster. The secondary metabolites present in D. ocellatus can be considered as a bona fide candidate in novel drug development to target reproductive diseases in which oxidative stress is involved. Moreover, it can be concluded that D. melanogaster is an ideal model organism to induce cellular stress in vitro and study therapeutic potential of lichen extracts.

Limitation of food availability (starvation) is known to influence the reproductive ability of animals. Autophagy is a lysosomal driven degradation process that protects the cell under metabolic stress conditions, such as during nutrient shortage. Whether, and how starvation-induced autophagy impacts on the maturation and function of reproductive organs in animals are still open questions. In this study, we have investigated the effects of starvation on histological and cellular changes that may be associated with autophagy in the ovary of the giant freshwater prawn, Macrobachium rosenbergii. To this end, the female prawns were daily fed (controls) or unfed (starvation condition) for up to 12 days, and the ovary tissue was analyzed at different time-points. Starvation triggered ovarian maturation, and concomitantly increased the expression of autophagy markers in vitellogenic oocytes. The immunoreactivities for autophagy markers, including Beclin1, LC3-II, and Lamp1, were enhanced in the late oocytes within the mature ovaries, especially at the vitellogenic stages. These markers co-localized with vitellin in the yolk granules within the oocytes, suggesting that autophagy induced by starvation could drive vitellin utilization, thus promoting ovarian maturation.