Asprosin-mediated regulation of ovarian functions in mice: An age-dependent study

Neo-oogenesis: Has its existence been proven?

The possible presence of oocyte and granulosa cells originated from stem cells in the adult mammalian ovaries was claimed by some studies which will lead to major changes in reproductive biology and infertility treatments. Purpose of this research is to investigate the possible existence and the location of the potential stem cells in mouse ovaries. In this study, the ovaries from 2-week (pre-puberty) and 8-week (adult) old BALB-C mice were used. For the investigation of the presence of possible stem cells, the expression profiles of three well known stem cell markers, Oct-4, Nanog and Sox2 were determined in the ovaries of two different age groups by real time quantitative RT-PCR (qRT-PCR). Protein expression levels and their localization in the ovary cells were immunohistochemically evaluated on fresh-frozen ovary tissue sections by using monoclonal antibodies specific to Sox2, Nanog and Oct-4. The gene expression levels of Oct-4 and Nanog were found to be significantly differentiated between 2-week old and 8-week old mice whereas no significant difference was observed in the expression level of Sox2 between two age groups. Immunohistochemistry results showed the presence of both Sox2 and Oct-4 protein in the cytoplasm of ovarian epithelial cells, granulosa cells, oocytes and theca cells. Nanog protein was observed only in the nucleus of the oocytes and furthermore the expression of Nanog was higher in eight weeks old samples compared to two weeks old ones according to qRT-PCR results. These results suggest for the first time that Nanog protein is expressed both in adult and pre-puberty mouse ovaries and locate at the nucleus of the oocytes and to the best of our knowledge this is the first study that shows the differential expression of Oct-4, Nanog and Sox2 in pre-puberty and adult mouse ovaries by qRT-PCR. Collectively, our results may suggest that both pre-puberty and adult mice ovaries accommodate cells carrying stem cell features.

There is no neo-oogenesis in the adult mammalian ovary

Contemporary claims that mitotically active female germ line or oogonial stem cells (OSCs) exist and support oogenesis during postnatal life in mammals have been debated in the field of reproductive biology since March 2004, when a mouse study posed the first serious challenge to the dogma of a fixed pool of oocytes being endowed at birth in more than 50 years. Other studies have since been put forth that further question the validity of this dogma, including the isolation of OSCs from neonatal and adult mouse ovaries by 4 independent groups using multiple strategies. Two of these groups also reported that isolated mouse OSCs, once transplanted back into ovaries of adult female mice, differentiate into fully functional eggs that ovulate, fertilize, and produce healthy embryos and offspring. Arguably, one of the most significant advances in this emerging field was provided by a new research study published this year, which reported the successful isolation and functional characterization of OSCs from ovaries of reproductive age women. Two commentaries on this latest work, one cautiously supportive and one highly skeptical, were published soon afterward. This article evaluates the current literature regarding postnatal oogenesis in mammals and discusses important next steps for future work on OSC biology and function.

Wide application of gonadotropin-releasing hormone (GnRH) agonists and antagonists for clinical purposes determines their effects on ovarian signaling pathways. Our study aimed to determine the localization, expression levels of Wnt signaling members in the pubertal and adult mouse ovary and the impact of GnRH antagonist cetrorelix on these signaling members. 0.5mg/kg of cetrorelix was injected to 3-and 6-week-old mice for 2 weeks. At the end of injection, ovaries from 5 (5Ce)- to 8-week (8Ce)-old mice were embedded in paraffin for immunohistochemistry and homogenized for western blot to compare with control (5C-8C) and sham groups (5S-8S). WNT2 and WNT4 showed higher expression in thecal and stromal cells in adult mouse ovaries and only WNT4 expression was affected by cetrorelix. FZD1 was localized mainly in oocytes of pubertal ovaries and granulosa cells and oocytes of adult ovaries. FZD1 was reduced by cetrorelix in pubertal ovaries. FZD4 was abundantly localized in thecal and stromal cells of all groups and protein level was not affected by cetrorelix. LRP-6 was expressed mainly in oocytes and stromal cells of pubertal, oocytes of adult ovaries and its expression was reduced by cetrorelix in adult ovaries. CTNNB1 intensity in granulosa cells was the lowest in pubertal and the highest in adult ovaries and its expression was decreased by cetrorelix in adult ovaries. Cetrorelix affected the expression of specific members of the Wnt signaling depending on the developmental stage of mice, pointing out its possible interaction with gonadotropins during pubertal and adult stages.

Perfluorooctanoic acid (PFOA) is a synthetic fluorosurfactant used in the manufacturing of fluorotelomers. Although PFOA is no longer produced in the United States, it is environmentally persistent and found in imported food packaging, cookware, and textiles. Previous studies have identified developmental toxicity of PFOA, but little is known about the effects of PFOA on the adult ovary. Thus, this study examined the effects of PFOA on hormone levels, ovarian steroidogenic gene expression, and folliculogenesis in mice in vitro and in vivo. For the in vitro studies, antral follicles from adult female mice were cultured with vehicle control or 1, 10, or 100 μg/ml PFOA for 96 h. For the in vivo studies, adult CD-1 female mice were orally dosed with vehicle control or 1, 5, 10, or 20 mg/kg/day PFOA for 10 days. Gene expression of steroidogenic enzymes, levels of sex steroid hormones, and follicle counts were analyzed. In vitro, PFOA (100 μg/ml) significantly decreased follicle growth, estradiol and estrone levels, and gene expression of StaR, Cyp11a1, and Hsd3b1 compared with controls. In vivo, exposure to PFOA significantly decreased progesterone and pregnenolone levels (5 mg/kg), increased testosterone levels (1 mg/kg), and increased gene expression of Cyp19a1 (1 mg/kg) compared with controls. Exposure to PFOA also significantly altered follicle counts by decreasing primordial follicles and increasing preantral and antral follicles (5 and 10 mg/kg) compared with controls. Collectively, these data show that PFOA disrupts adult ovarian function in a nonmonotonic matter and may pose a risk for premature ovarian failure.

Lately, stem cell approaches have provided new information on reproductive organ function and additionally recommended novel treatment possibilities. The type(s) and differentiation potential of stem cells present in the mammalian ovary are largely unknown; while oogonial stem cells have been reported, we explored the possibility that multipotent stem cells may reside in the ovary and have wide differentiation potential. In this experimental study, homogenates of whole mouse ovaries were sorted using the stem cell surface markers stem cell antigen-1 and stage specific embryonic antigen-1/CD15. Viable double-positive cells 3-10μm in diameter were evaluated immediately after sorting and after culture using differentiation conditions. Ovarian-derived stem cells were differentiated into the three main cell types: adipocytes, chondrocytes, or osteocytes. The subsequent culture was performed in media containing bone morphogenetic protein 4 (BMP-4) and/or retinoic acid (RA). RA, BMP-4 or the two agents in combination, consistently stimulated germ cell gene expression. RA treatment strongly stimulated germline gene expression and also the development of cells that were morphologically reminiscent of oocytes. The germ cell genes Dazl, Ddx4, Figla, Gdf-9, Nobox, Prdm9, and Sycp-1 were all detected at low levels. Remarkably, treatment with BMP-4 alone significantly increased protein expression of the granulosa cell product anti-Müllerian hormone (AMH). We have shown that an inclusive isolation protocol results in the consistent derivation of multipotent stem cells from the adult ovary; these cells can be differentiated towards the germ cell fate (RA alone), somatic ovarian cell fate as indicated by AMH production (BMP-4 alone), or classical mesenchymal cell types. Taken together, these data suggest the presence of multipotent mesenchymal stem cells in the murine ovary.

Understanding the molecular pathways that underpin ovarian development and function is vital for improving the research approaches to investigating fertility. Despite a significant improvement in our knowledge of molecular activity in the ovary, many questions remain unanswered in the quest to understand factors influencing fertility and ovarian pathologies such as cancer. Here, we present an investigation into the expression and function of the developmental transcription factor LIM Homeobox 9 (LHX9) in the adult mouse ovary. We have characterized Lhx9 expression in several cell types of the mature ovary across follicle stages. To evaluate possible LHX9 function in the adult ovary, we investigated ovarian anatomy and transcription in an Lhx9+/- knockout mouse model displaying subfertility. Despite a lack of gross anatomical differences between genotypes, RNA-sequencing found that 90 differentially expressed genes between Lhx9+/ - and Lhx9+/+ mice. Gene ontology analyses revealed a reduced expression of genes with major roles in ovarian steroidogenesis and an increased expression of genes associated with ovarian cancer. Analysis of the ovarian epithelium revealed Lhx9+/ - mice have a disorganized epithelial phenotype, corresponding to a significant increase in epithelial marker gene expression. These results provide an analysis of Lhx9 in the adult mouse ovary, suggesting a role in fertility and ovarian epithelial cancer.

An unlimited supply of ovarian follicles / oocytes could potentially mean a slowing down of a woman's biological clock. This avant-garde has come from new research in stem cells that has challenged the dogma that females are born with fixed number of eggs, which exhaust during her approximately 30 reproductive years. The human ovum-producing stem cell now discovered in the ovaries of reproductive-age women, can generate oocytes in-vitro and in vivo. 1 Embryonic Stem (ES) cells research has been the most controversial and debatable since they were first isolated from mouse in 1981. 2 ES cells possess the potential of producing an endogenous specialized tissue and the possibility of organ engineering raises ethical concerns regarding instrumental use of embryos for malpractices. The earliest challenge to the theory of an exhaustible number of oocytes came in 2004, when Johnson and coworkers discovered that juvenile and adult mouse ovaries possess mitotically active germ cells.3 These cells continuously replenish the follicle pool in adult reproductive female mice and express the meiotic entry marker (synaptonemal complex protein 3) on their surface. Also treatment with busulfan, a mitotic germ-cell toxicant, reduce the primordial follicle reserve resulting in an earlier onset of menopause in these mice. This discovery established the existence of female germline stem cells in juvenile and adult mice.

Oocyte Family Trees: Old Branches or New Stems?

O-25: FOXL2, a gene associated with premature ovarian failure, interacts with large tumor suppressor gene 1 (LATS1)

Female germline or oogonial stem cells transiently residing in fetal ovaries are analogous to the spermatogonial stem cells or germline stem cells (GSCs) in adult testes where GSCs and meiosis continuously renew. Oocytes can be generated in vitro from embryonic stem cells and induced pluripotent stem cells, but the existence of GSCs and neo-oogenesis in adult mammalian ovaries is less clear. Preliminary findings of GSCs and neo-oogenesis in mice and humans have not been consistently reproducible. Monkeys provide the most relevant model of human ovarian biology. We searched for GSCs and neo-meiosis in ovaries of adult monkeys at various ages, and compared them with GSCs from adult monkey testis, which are characterized by cytoplasmic staining for the germ cell marker DAZL and nuclear expression of the proliferative markers PCNA and KI67, and pluripotency-associated genes LIN28 and SOX2, and lack of nuclear LAMIN A, a marker for cell differentiation. Early meiocytes undergo homologous pairing at prophase I distinguished by synaptonemal complex lateral filaments with telomere perinuclear distribution. By exhaustive searching using comprehensive experimental approaches, we show that proliferative GSCs and neo-meiocytes by these specific criteria were undetectable in adult mouse and monkey ovaries. However, we found proliferative nongermline somatic stem cells that do not express LAMIN A and germ cell markers in the adult ovaries, notably in the cortex and granulosa cells of growing follicles. These data support the paradigm that adult ovaries do not undergo germ cell renewal, which may contribute significantly to ovarian senescence that occurs with age.

Effects of pair-housing pubertal and adult male and female mice on LPS-induced age-dependent immune responses: A potential role for the gut microbiota

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that can regulate the expression of complementary mRNA targets. Identifying tissue-specific miRNAs is the first step toward understanding the biological functions of miRNAs, which include the regulation of tissue differentiation and the maintenance of tissue identity. In this study, we performed small RNA library sequencing in adult mouse testis and ovary to reveal their characteristic organ- and gender-specific profiles and to elucidate the characteristics of the miRNAs expressed in the reproductive system. We obtained 10,852 and 11 744 small RNA clones from mouse testis and ovary respectively (greater than 10,000 clones per organ), which included 6630 (159 genes) and 10,192 (154 genes) known miRNAs. A high level of efficiency of miRNA library sequencing was achieved: 61% (6630 miRNA clones/10,852 small RNA clones) and 87% (10,192/11,744) for adult mouse testis and ovary respectively. We obtained characteristic miRNA signatures in testis and ovary; 55 miRNAs were detected highly, exclusively, or predominantly in adult mouse testis and ovary, and discovered two novel miRNAs. Male-biased expression of miRNAs occurred on the X-chromosome. Our data provide important information on sex differences in miRNA expression that should facilitate studies of the reproductive organ-specific roles of miRNAs.

Electrical synapses formed by gap junctions containing connexin36 (Cx36) promote synchronous activity of interneurones in many regions of mammalian brain; however, there is limited information on the role of electrical synapses in spinal neuronal networks. Here we show that Cx36 is widely distributed in the spinal cord and is involved in mechanisms that govern presynaptic inhibition of primary afferent terminals. Electrophysiological recordings were made in spinal cord preparations from 8- to 11-day-old wild-type and Cx36 knockout mice. Several features associated with presynaptic inhibition evoked by conditioning stimulation of low threshold hindlimb afferents were substantially compromised in Cx36 knockout mice. Dorsal root potentials (DRPs) evoked by low intensity stimulation of sensory afferents were reduced in amplitude by 79% and in duration by 67% in Cx36 knockouts. DRPs were similarly affected in wild-types by bath application of gap junction blockers. Consistent with presynaptic inhibition of group Ia muscle spindle afferent terminals on motoneurones described in adult cats, conditioning stimulation of an adjacent dorsal root evoked a long duration inhibition of monosynaptic reflexes recorded from the ventral root in wild-type mice, and this inhibition was antagonized by bicuculline. The same conditioning stimulation failed to inhibit monosynaptic reflexes in Cx36 knockout mice. Immunofluorescence labelling for Cx36 was found throughout the dorsal and ventral horns of the spinal cord of juvenile mice and persisted in mature animals. In deep dorsal horn laminae, where interneurones involved in presynaptic inhibition of large diameter muscle afferents are located, cells were extensively dye-coupled following intracellular neurobiotin injection. Coupled cells displayed Cx36-positive puncta along their processes. Our results indicate that gap junctions formed by Cx36 in spinal cord are required for maintenance of presynaptic inhibition, including the regulation of transmission from Ia muscle spindle afferents. In addition to a role in presynaptic inhibition in juvenile animals, the persistence of Cx36 expression among spinal neuronal populations in the adult mouse suggests that the contribution of electrical synapses to integrative processes in fully mature spinal cord may be as diverse as that found in other areas of the CNS.