The organization of the female reproductive system in the parasitic mite Myobia murismusculi (Trombidiformes: Myobiidae)

The general morphology of the internal genital organs in Myobia murismusculi females have been studied by means of light and electron microscopy. It is shown, that their reproductive system is composed of the single ovary, long complex oviduct, cuticular vagina and receptaculum seminis. The apical region of the ovary is formed by the numerous processes of gonadian somatic cells. These cells generate the ovarian sheath and probably take place in oocyte's metabolism. Any nutritive cells in the Myobia ovary are absent. The vitellogenetic oocytes develop in the ovarian pouches like in other trombidiform mites. The oviduct consists of three parts, each of them has a specific ultrastructure and type of secretory product. Two proximal parts of the oviduct produce the egg-shell components, while the third distal part never contains oocytes. It could be suggested that this part produces a special secret onto the oocyte surface to glue the oocytes to the hairs of host.     

Gonadal development and diandric protogyny in two populations of Dascyllus reticulatus from Madang, Papua New Guinea

Two Dascyllus reticulatus populations from Madang, Papua New Guinea exhibited diandric protogyny. In both populations, gonads began as undifferentiated, and then developed oocytes in the primary growth stage and an ovarian lumen. From this ovarian state or from more developed ovaries containing oocytes beyond the primary‐growth stage, some gonads developed into testes. The first sign of testicular development was degeneration of oocytes, degeneration of oocytes in the primary growth stage in ovarian gonads and degeneration of oocytes of all growth stages present including the primary growth stage in ovaries, which was then followed by development of spermatogenic tissue. In both populations, most of the fish that had gonads with degenerating oocytes were smaller than the smallest mature females, indicating that development towards testes was mostly initiated in immature gonads containing only pre‐vitellogenic oocytes. On some occasions, however, females as large as other mature females also had gonads with degenerating oocytes, suggesting that development towards testes may have occurred in mature ovaries as well. This latter notion is further strengthened by the discovery of a fish having a gonad that contained both degenerating vitellogenic oocytes and developing spermatogenic tissue. Taken together, these results suggest that D. reticulatus can exhibit diandric protogyny, because testes in D. reticulatus developed from juvenile gonads as well as from mature ovaries.     

The ovarian follicle as a model for the cell-mediated control of tissue growth

Summary Thy-1⁺ cells, producing Thy-1⁺ material, have been demonstrated by the indirect immunoperoxidase technique in the theca of growing ovarian follicles of the rat. OX-2 antigen, known as the minor glycoprotein of rat thymocytes, was detected in granulosa cells of non-growing follicles. Ia⁺ cells of dendritic type and/or activated macrophages were identified in the granulosa of advanced degenerating follicles, and remnants of the zona pellucida exhibited immunoglobulins. In some ovaries immunoglobulins were also bound to the zona pellucida of oocytes of early degenerating antral follicles. Medium-sized antral follicles with degenerating granulosa were occasionally invaded by cells carrying antigens of cytotoxic T lymphocytes or other T lymphocyte subsets, while degenerating large antral follicles were sometimes invaded by cells exhibiting antigen of cells with natural killer function (but not antigens of T lymphocytes). Granulosa cells of some degenerating antral follicles exhibited class-I antigens derived from the major histocompatibility complex. We suggest that cell-mediated control mechanisms of antigen expression and metabolism of tissue cells during their differentiation and degeneration should be considered in addition to the well-documented hormonal dependence of some tissues.     

Gonadal development and non-functional protogyny in a coral-reef damselfish, Dascyllus albisella Gill

All gonads of the Hawaiian dascyllus Dascyllus albisella , irrespective of the final sex of individuals, developed an ovarian lumen and primary-growth-stage oocytes after an initially undifferentiated state. From this ovarian state or from more differentiated ovaries, some gonads redifferentiated into testes. None of 117 individuals examined had a gonad containing degenerating vitellogenic oocytes and proliferating spermatogenic tissue. Eleven individuals had gonads containing degenerating cortical-alveolus-stage oocytes and developing spermatogenic tissue. The size of these individuals overlapped with the female size range in which the majority of the females were still in the middle of the maturation process. They were absent from the larger size range where the majority of females had vitellogenic oocytes. This indicated that the transition toward maleness is likely to have occurred after the onset of cortical-alveolus stage, but before final oocyte maturation and spawning as females. Therefore the protogynous pattern of gonadal development was non-functional. There was no dimorphism in the sperm duct configuration, and all the testes were secondary testes reported for diandric, protogynous species with undelimited gonads. Very early development of an ovarian lumen appeared to have resulted in a secondary-male configuration in all testes, although redifferentiation into males appeared to have occurred before sexual maturity and spawning as females.     

Degeneration of germ line cells in amphibian ovary

Ogielska, M., Rozenblut, B., Augustyńska, R., Kotusz, A. 2010. Degeneration of germ line cells in amphibian ovary. —Acta Zoologica (Stockholm) 91 : 319–327 We studied the morphology of degenerating ovarian follicles in juvenile and adult frogs Rana temporaria, Rana lessonae and Rana ridibunda. Degeneration of primordial germ cells was never observed and was extremely rare in oogonia and early oocytes in a cyst phase in juveniles. Previtellogenic oocytes were rarely affected. Three main types of atresia were identified. In type I (subdivided into stages A–D), vitellogenic oocytes are digested by proliferating follicle cells that hypertrophy and become phagocytic. A – germinal vesicle shrinks, nucleoli fuse, oocyte envelope interrupts, and follicular cells hypertrophy; B – follicular cells multiply and invade the oocyte; C – entire vesicle is filled by phagocytic cells; D – degenerating phagocytes accumulate black pigment. Type II is rare and resembles breakdown of follicles and release of ooplasm. In type III, observed in previtellogenic and early vitellogenic oocytes, ooplasm and germinal vesicle shrink, follicle cells do not invade the vesicle, and condensed ooplasm becomes fragmented. The residual oogonia in adult ovaries (germ patches) multiply, but soon degenerate.     

Co-expression of the SARS-CoV-2 entry molecules ACE2 and TMPRSS2 in human ovaries: Identification of cell types and trends with age

The high rate of SARS-CoV-2 infection poses a serious threat to public health. Previous studies have suggested that SARS-CoV-2 can infect human ovary, the core organ of the female reproductive system. However, it remains unclear which type of ovarian cells are easily infected by SARS-CoV-2 and whether ovarian infectivity differs from puberty to menopause. In this study, public datasets containing bulk and single-cell RNA-Seq data derived from ovarian tissues were analyzed to demonstrate the mRNA expression and protein distribution of the two key entry receptors for SARS-CoV-2—angiotensin-converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2). Furthermore, an immunohistochemical study of ACE2 and TMPRSS2 in human ovaries of different ages was conducted. Differentially expressed gene (DEG) analysis of ovaries of different ages and with varying ovarian reserves was conducted to explore the potential functions of ACE2 and TMPRSS2 in the ovary. The analysis of the public datasets indicated that the co-expression of ACE2 and TMPRSS2 was observed mostly in oocytes and partially in granulosa cells. However, no marked difference was observed in ACE2 or TMPRSS2 expression between young and old ovaries and ovaries with low and high reserves. Correspondingly, ACE2 and TMPRSS2 were detected in the human ovarian cortex and medulla, especially in oocytes of different stages, with no observed variations in their expression level in ovaries of different ages, which was consistent with the results of bioinformatic analyses. Remarkably, DEG analysis showed that a series of viral infection-related pathways were more enriched in ACE2-positive ovarian cells than in ACE2-negative ovarian cells, suggesting that SARS-CoV-2 may potentially target specific ovarian cells and affect ovarian function. However, further fundamental and clinical research is still needed to monitor the process of SARS-CoV-2 entry into ovarian cells and the long-term effects of SARS-CoV-2 infection on the ovarian function in recovered females.     

Gonadal development and an indication of functional protogyny in the Indian damselfish (Dascyllus carneus)

The objective of this study was to determine the sexual pattern of the Indian dascyllus Dascyllus carneus . After an initially undifferentiated state, gonads of D. carneus developed an ovarian lumen and primary growth stage oocytes, and subsequently cortical-alveolus stage oocytes. From ovaries with cortical-alveolus stage oocytes and from more developed ovaries, some gonads redifferentiated into testes. From a sample of 163 individuals, two had a gonad containing degenerating vitellogenic oocytes and proliferating spermatogenic tissue, nine had a gonad containing degenerating cortical-alveolus stage oocytes and spermatogenic tissue, and five had a gonad with degenerating primary growth stage oocytes and spermatogenic tissue. The size of these individuals overlapped greatly with the size range of mature females, suggesting that at least in some individuals, redifferentiation toward a testis occurred after spawning as females. This indicates that D. carneus is a functional, diandric protogynous hermaphrodite. Removal of a dominant male(s) did not induce a sex change in any of the ranking females in the laboratory and field groups. There was no difference in the number of chases and signal jumps performed by the ranking female between control and experimental field groups, or before and after removal of the male. However, the sizes of the ranking females were at or beyond the size range of individuals with a mixed-stage gonad, suggesting that the developmental window for female-to-male sex change may not be open ended. In 41 of 43 field groups, in which sex of fish was determined histologically or by the shape of the urogenital papilla, one to several highest size ranks were occupied by males, followed by one to several females. Mature males, however, were not limited to the highest ranks and occurred at various lower size ranks within groups. Individuals with a mixed-stage gonad also occupied various size ranks within groups.     

Effects of delayed excision of oviducts/ovaries on mouse oocytes and embryos

To achieve the best and reproducible results of experiments, effects of delayed excision of oviducts/ovaries on mouse ovarian/ovulated oocytes and embryos have been studied. Oviducts/ovaries were excised at different times after death of mice and effects of the postmortem interval on ovarian/ovulated oocytes and embryos were analyzed. When oviduct excision was delayed 10 min, many ovulated oocytes lysed or underwent in vitro spontaneous activation, and this postmortem effect aggravated with the extension of postmortem interval and oocyte aging. Oocytes from different mouse strains responded differently to delayed oviduct removal. Delayed oviduct excision did not cause lysis of zygotes or embryos but compromised their developmental potential. When ovaries were excised at 30 min after death, percentages of atretic follicles increased while blastocyst cell number declined significantly after oocyte maturation in vitro. Preservation of oviducts in vitro, in intact or opened abdomen at different temperatures and histological analysis of oviducts from different treatments suggested that toxic substance(s) were secreted from the dying oviducts which induced oocyte lysis and spontaneous activation and both this effect itself and the sensitivity of oocytes to this effect was temperature dependent. It is concluded that a short delay of oviduct/ovary removal had marked detrimental effects on oocytes and embryos. This must be taken into account in experiments using oocytes or embryos from slaughtered animals. The data may also be important for estimation of the time of death in forensic medicine and for rescue of oocytes from deceased valuable or endangered mammals.     

The effect of nutrition and metabolic status on the development of follicles,oocytes and embryos in ruminants

The impact of nutrition and energy reserves on the fertility of ruminants has been extensively described. However, the metabolic factors and the molecular mechanisms involved in the interactions between nutrition and ovarian function are still poorly understood. These factors could be hormonal (either reproductive and/or metabolic) and/or dietary and metabolic (glucose, amino acids and fatty acids). In this review, we briefly summarize the impact of those nutrients (fatty acids, glucose and amino acids) and metabolic hormones (insulin/IGF-I, growth hormone, T3/4, ghrelin, apelin and the adipokines (leptin, adiponectin and resistin)) implicated in the development of ovarian follicles, oocytes and embryos in ruminants. We then discuss the current hypotheses on the mechanisms of action of these factors on ovarian function. We particularly describe the role of some energy sensors including adenosine monophosphate-activated kinase and peroxisome proliferator-activated receptors in the ovarian cells.     

Sexual maturation in female triploid plaice, Pleuronectes platessa, and plaice × flounder, Platichthys flesus, hybrids

The gonads of immature female triploid plaice ( Pleuronectes plafcssa ) and plaice × flounder ( Platichthys flesus ) hybrids produced from cold-shocked eggs were examined. They were less than half the size of those in diploids of similar age and contained only small numbers of developing oocytes. These were similar both in size and cytological appearance to those in the controls. The bulk of the ovarian tissue was composed of nests of small undifferentiated cells resembling the oogonia in the ovaries of diploid fish. Mature, 5 year old triploid hybrids which had not produced eggs over the previous two spawning seasons were killed in January when the diploid controls were nearing spawning condition. The ovaries of these fish were smaller than those of the controls and contained degenerating oocytes at maturation stage V. The advantage of sterile female triploid fish for fish-cultivation is discussed.     

Gonadal restructuring and correlative steroid hormone profiles during natural sex change in protogynous honeycomb grouper (Epinephelus merra)

The honeycomb grouper shows protogynous hermaphroditism. The endocrine mechanisms involved in gonadal restructuring throughout protogynous sex change are largely unknown. In the present study, we investigated changes in the gonadal structures and levels of serum sex steroid hormones during female to male sex change in the honeycomb grouper. On the basis of histological changes, entire process of sex change was assigned into four developmental phases: female, early transition (ET), late transition (LT), and male phase. At the female phase, the oocytes of several developmental stages were observed including gonial germ cells in the periphery of ovigerous lamellae. At the beginning of ET phase, perinucleolar and previtellogenic oocytes began degenerating, followed by proliferation of spermatogonia toward the center of lamella. The LT phase was characterized by further degeneration of oocytes and rapid proliferation of spermatogenic germ cells throughout the gonad. At the male phase, no ovarian cells were observed and testis had germ cells undergoing active spermatogenesis. Serum levels of estradiol-17beta (E2) were high in females in the breeding season, but low in the non-breeding female, transitional and male phase, and those of 11-ketotestosterone (11-KT) and testosterone (T) were low in females and gradually increased in the transitional and male phase. The present results suggest that low serum E2 levels and degeneration of oocytes accompanied by concomitant increase in the 11-KT levels and proliferation of spermatogenic germ cells are probably the events mediating protogynous sex change in the honeycomb grouper.     

Ovarian germinal epithelium and folliculogenesis in the common snook, Centropomus undecimalis (Teleostei: centropomidae)

The ovarian germinal epithelium in the common snook, Centropomus undecimalis, is described. It consists of epithelial and prefollicle cells that surround germ cells, either oogonia or oocytes, respectively. The germinal epithelium borders a body cavity, the ovarian lumen, and is supported by a basement membrane that also separates the epithelial compartment of the ovarian lamellae from the stromal compartment. During folliculogenesis, the epithelial cells, whose cytoplasmic processes encompass meiotic oocytes, transform into prefollicle cells, which become follicle cells at the completion of folliculogenesis. The follicle is a derivative of the germinal epithelium and is composed of the oocyte and surrounding follicle cells. It is separated from the encompassing theca by a basement membrane. The cells that form the theca interna are derived from prethecal cells within the extravascular space of the ovarian stroma. The theca externa differentiates from undifferentiated cells within the stromal compartment of the ovary, from within the extravascular space. The theca interna and the theca externa are not considered to be part of the follicle and are derived from a different ovarian compartment than the follicle. Meiosis commences while oocytes are still within the germinal epithelium and proceeds as far as arrested diplotene of the first meiotic prophase. The primary growth phase of oocyte development also begins while oocytes are still within the germinal epithelium or attached to it in a cell nest. The definitions used herein are consistent between sexes and with the mammalian literature.     

Can ovarian infertility be treated with bone marrow- or ovary-derived germ cells?

A year ago, reproductive biologists and general public were astonished with evidence reported by Johnson et al. in Nature 428:145 that mammalian ovaries possess persisting large germline stem cells, which allegedly enable follicular renewal in adult females. Recently, the same research group declared such view obscure, and reported that mammalian oocytes originate from putative germ cells in bone marrow and are distributed by peripheral blood to the ovaries (Cell 122:303). While neglecting available data on the germ cell origin from the ovarian surface epithelium (OSE) in adult mouse and human females and complexity of follicular renewal in humans, the authors widely extrapolated their observations on formation of allogeneic oocytes after bone marrow (or blood) transplantation in ovaries of adult mice treated with cytostatics to clinical implications in the public media. Yet, the resulting outcome that such allogeneic oocytes may enable the propagation of ovarian cycles is a poor alleviation for the women with ovarian infertility. Women lacking primary follicles, or carrying follicles with low quality eggs persisting in aging ovaries, are not concerned about the lack of menstrual cycles or ovarian steroids, but about virtually no chance of having genetically related children. Johnson et al. also reported that the germ cell formation in bone marrow disappears in ovariectomized mice. Such observation, however, raises solid doubts on the bone marrow origin of oocytes. Since germ cells developing from the OSE cells of adult human ovaries during periodical follicular renewal are known to enter blood vessels in order to enable formation of primary follicles at distant ovarian sites, they also contaminate peripheral blood and hence bone marrow. Better knowledge on the complexity of follicular renewal in humans and exploration of a potential of human OSE cells to produce new oocytes in vitro are essential for novel approaches to the autologous treatment of premature ovarian failure and age induced ovarian infertility.     

Über die Degeneration von Eizellen des Zebrafisches,Brachydanio rerio,in vitro

Zusammenfassung In Ovarkulturen des Zebrafisches, Brachydanio rerio, wird der Degenerationsverlauf der Eizellen verschiedener Entwicklungsstadien beschrieben. Junge dotterlose Oocyten überstehen die 4 wöchige Kultivierung ohne wesentliche morphologische oder histochemisch nachweisbare Veränderungen. Oocyten mittleren Alters, die bereits Rindenvakuolen enthalten, verlieren im Laufe der Kultivierung ihre cytoplasmatische Basophilie und ihre in vivo zahlreichen Nukleolen. Bei einem Teil dieser Eizellen sind als Zeichen beginnender Degeneration Follikelepithelzellen im randständigen Cytoplasma eingewandert. Reife Oocyten beginnen nach 1–2 Wochen mit der Degeneration. Das in der Kultur auswachsende Follikelepithel behält seine Fähigkeit bei, in degenerierende Eizellen einzuwandern und Dotter zu phagocytieren.

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Oocyte degeneration in the zebrafish, Brachydanio rerio, in vitro

Summary In cultured ovarian tissue of the zebrafish, Brachydanio rerio, the degeneration of oocytes in various developmental stages is described. Young yolk-free oocytes survive the 4 weeks cultivation time without significant morphological and histochemical alterations. In medium sized oocytes, which contain cortical granules already, the cytoplasmic basophilia and the number of nucleoli decrease in the culture. Some of these oocytes show follicular epithelial cells invading in the peripheral cytoplasm. Mature oocytes become atretic after 1 or 2 weeks cultivation time. The outgrowing follicular epithelial cells retain their ability to penetrate degenerating oocytes and to take up yolk by means of phagocytosis.

     

Mitochondrial aggregation patterns and activity in in vitro cultured bovine oocytes recovered from early antral ovarian follicles

The low developmental competence seen in in vitro cultured oocytes collected from early antral follicles may be related to their mitochondrial status. The aim of this study was to examine the chromatin configuration, pattern of mitochondrial aggregation and mitochondrial activity of non-cultured and in vitro-cultured bovine oocytes originating from early antral ovarian follicles. Cumulus-oocyte complexes with adjacent granulosa cells (COCGs) were recovered from early antral follicles of 0.4 to 0.8 mm diameter. Control (Day 0) oocytes were recovered from freshly collected COCGs and fixed and stained. Selected COCGs were placed in growth culture for 7 days (Day 7) or 14 days (Day 14). Following growth culture, COCs with normal appearance were placed in maturation medium (IVM) for 24 h and then fixed and stained with MitoTracker CMTM Ros Orange and Hoechst 33258. The percentage of oocytes with an immature meiotic configuration after growth culture decreased with the time of growth culture, being 96.7; 72.5 and 35.4% respectively for Day 0, Day 7 and Day 14 of culture; the remaining oocytes were degenerating or resuming meiosis. After subsequent IVM the highest proportion of oocytes in diakinesis or metaphase I was found in the D7+IVM group (59.4%). When growth culture was prolonged to day 14 and IVM, the number of degenerated oocytes increased dramatically after IVM. The mitochondrial distribution in the oocytes changed from homogeneous to heterogeneous as growth culture time increased. The respiratory activity as measured by fluorescence intensity increased over the time of growth culture, and was highest in oocytes that had resumed GVBD. In conclusion, for oocytes in isolated COCGs from early antral follicles, culture conditions longer than 7 days should be more adapted for a slow nuclear maturation accompanied by a decreased energy metabolism to prevent chromatin pycnosis.     

Follicular atresia in relation to oocyte morphology in non-pregnant and pregnant women

Antral follicles from normally menstruating women and women in the third trimester of pregnancy were classified as healthy or atretic by flow cytometric DNA measurements on aspirated granulosa cells and by the concentration of steroids in the follicular fluid. The oocytes contained in these follicles were characterized as healthy or degenerating by their morphology at the light microscopic level. In 98% of the cases (61/62) morphologically healthy or degenerating oocytes were found in follicles which were classified as healthy or atretic respectively. In the normally menstruating women degenerative changes in the oocyte and the remainder of the follicle appeared to occur in synchrony. In pregnant women asynchrony was noted between the oocyte and the remainder of the follicle as follicular atresia progressed. The present study demonstrated the usefulness of flow cytometric DNA measurement in characterizing antral follicles of all sizes as healthy or more or less atretic.     

Oogenesis in cultures derived from adult human ovaries

Ten years ago, we reported that in adult human females the ovarian surface epithelium (OSE) is a source of germ cells. Recently, we also demonstrated that new primary follicles are formed by assembly of oocytes with nests of primitive granulosa cells in the ovarian cortex. The components of the new primary follicles, primitive granulosa and germ cells, differentiated sequentially from the OSE, which arises from cytokeratin positive mesenchymal progenitor cells residing in the ovarian tunica albuginea. In the present study, we investigated the possibility that the oocytes and granulosa cells may differentiate in cultures derived from adult human ovaries. Cells were scrapped from the surface of ovaries and cultured for 5 to 6 days, in the presence or absence of estrogenic stimuli [phenol red (PhR)]. The OSE cells cultured in the medium without PhR differentiated into small (15 micron) cells of granulosa phenotype, and epithelial, neural, and mesenchymal type cells. In contrast, OSE cells cultured in the presence of PhR differentiated directly into large (180 micron) cells of the oocyte phenotype. Such cells exhibited germinal vesicle breakdown, expulsion of the polar body, and surface expression of zona pellucida proteins, i.e. characteristics of secondary oocytes. These in vitro studies confirm our in vivo observations that in adult human ovaries, the OSE is a bipotent source of oocytes and granulosa cells. Development of numerous mature oocytes from adult ovarian stem cells in vitro offers new strategies for the egg preservation, IVF utilization, and treatment of female infertility. In addition, other clinical applications aiming to utilize stem cells, and basic stem cell research as well, may employ totipotent embryonic stem cells developing from fertilized oocytes.