Lecture 8: Stem cells and female fertility

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What is the primary endpoint of the in vitro maturation process described?

Culturing for more than 16 hours

Attainment of oocytes at the metaphase I stage

Generation of phenotypically normal offspring (correct)

Development of germinal vesicle oocytes

How long did it take for the complete process from stem cell to fertilizable mouse oocyte?

11 months

16 hours

4 weeks

33 days (correct)

At which stage are oocytes ready for ovulation after in vitro maturation?

Metaphase II (correct)

Germinal vesicle stage

Prophase I

Metaphase I

What type of analysis was performed on the imprinted loci?

Epigenetic analysis Signup and view all the answers

What was the condition under which cumulus-oocyte complexes were cultured during in vitro maturation?

For 16 hours in standard IVM conditions Signup and view all the answers

What is suggested by the failure to detect meiotic markers in adult ovaries?

There may not be new oocyte formation from OSCs. Signup and view all the answers

What do parabiosis studies indicate about the origin of OSCs?

OSCs are formed from non-circulating germ cells. Signup and view all the answers

What issue is highlighted by the difficulty other researchers have had in replicating Johnson et al.'s findings?

The original methods may be flawed. Signup and view all the answers

Which meiotic markers have studies failed to detect in adult ovaries?

STRA8, SCP3, Spo11, DMC1 Signup and view all the answers

What misconception does the finding of the absence of meiotic markers challenge?

Meiosis is a continuous process in adult female mice. Signup and view all the answers

What was the background objective of the 2006 study involving parabiosis?

To investigate the contribution of bone marrow-derived germ cells to oocyte formation. Signup and view all the answers

In the study referenced, what characteristic did the transgenic mouse express?

GFP (Green Fluorescent Protein). Signup and view all the answers

What marker is used to identify oogonial stem cells (OSCs) in the study?

DDX4 Signup and view all the answers

Which method is used for profiling the transcriptomes of the ovarian cortex cells?

10X Genomics Platform Signup and view all the answers

What type of tissue was primarily analyzed for the presence of primordial follicles?

Ovarian cortex Signup and view all the answers

What is the first step in processing the ovarian tissue for cellular analysis?

Dissection and Preservation Signup and view all the answers

After thawing the ovarian tissue, what was primarily removed from the cell suspensions?

Primordial follicles Signup and view all the answers

Which technology was utilized to analyze the transcriptomes of cultured cells?

SmartSeq2 Technology Signup and view all the answers

What was a notable outcome of the cellular analysis performed in the study?

Identification of over 24,000 cellular types Signup and view all the answers

Which process follows the removal of the medullary region in tissue processing?

Chopping and freezing cortex pieces Signup and view all the answers

What analysis method was used to investigate specific surface markers in the ovarian cells?

Fluorescence-Activated Cell Sorting (FACS) Signup and view all the answers

What is the purpose of transcriptomic profiling in this research?

To analyze different cell types present Signup and view all the answers

What was the primary objective of the parabiosis experiment involving the transgenic and non-transgenic mice?

To assess whether circulating germ cells contribute to oocyte production in the ovary. Signup and view all the answers

Which method was employed to visualize the cells derived from the transgenic mouse during the study?

Green fluorescent protein (GFP) expression to visualize cells. Signup and view all the answers

What was the outcome expected if the hypothesis regarding germ cell contribution was true?

The non-GFP mouse would ovulate GFP+ oocytes. Signup and view all the answers

What treatment did the non-transgenic mice receive to deplete their ovarian follicle reserves?

Cyclophosphamide and Busulfan to remove follicles. Signup and view all the answers

What hypothesis was being tested regarding the ovarian reserve and OSCs?

OSCs are activated only under conditions of ovarian reserve depletion. Signup and view all the answers

What was the significance of using both transgenic and non-transgenic mice in the study?

To explore potential contributions of circulating germ cells to oocyte formation. Signup and view all the answers

What does the presence of GFP+ oocytes in the non-GFP mouse potentially indicate?

Germ cells migrated from the transgenic mouse to the non-transgenic mouse. Signup and view all the answers

What was a key feature of the parabiosis setup employed in the study?

Surgical joining of mice to create a shared circulatory system. Signup and view all the answers

What was the purpose of inducing superovulation in the mice post-parabiosis?

To increase the likelihood of observing GFP+ oocyte production. Signup and view all the answers

What was the result regarding the origin of oocytes in GFP+ and non-GFP mice?

Only host-derived oocytes were found in both mice types. Signup and view all the answers

What conclusion can be drawn regarding bone marrow-derived oogonial stem cells?

They do not contribute to forming new oocytes post depletion. Signup and view all the answers

In the study analyzing the human ovarian cortex, what advanced methodology was utilized?

Single-cell RNA sequencing. Signup and view all the answers

What was the primary focus of the research conducted on the human ovarian cortex?

To assess the presence of potential oogonial stem cells. Signup and view all the answers

What experimental condition was used in the non-transgenic mice regarding follicle reserves?

Their follicle reserves were depleted. Signup and view all the answers

What evidence was found concerning circulating germ cells in non-transgenic mice?

No GFP+ oocytes were recovered. Signup and view all the answers

From which patient demographic were the ovarian cortex samples obtained for the study?

Individuals undergoing sex change procedures. Signup and view all the answers

What overall conclusion did the study provide regarding bone marrow-derived germ cells?

They do not contribute to oocyte formation in adult mice. Signup and view all the answers

What type of oocytes did the GFP+ mice specifically ovulate?

Only GFP+ oocytes. Signup and view all the answers

What was a key feature of the study's methodology regarding ovarian samples?

Samples were frozen for later analysis. Signup and view all the answers

Study Notes

Oogonial Stem Cells (OSCs)

OSCs are also known as egg precursor cells or female germline stem cells
They are present in the ovary after birth
They can proliferate and generate new oocytes
Their existence in the mammalian ovary is still debated and not confirmed

Controversy Surrounding OSCs

There is ongoing debate about their existence and whether they contribute to the ovarian reserve
Research on OSCs has triggered significant research
Many researchers remain skeptical about their existence

Evidence for OSCs

Studies claim that adult mouse ovaries contain stem cells capable of generating new eggs
This would allow for viable offspring to be produced
They aimed to investigate whether adult mouse ovaries contain stem cells that can generate new eggs throughout reproductive life

Evidence Against OSCs

Mathematical modeling and follicle counting suggest the sufficient number of oocytes/follicles at birth to last a lifetime
The presence of menopause contradicts the idea of continuous follicle renewal
Research in transgenic mice shows no post-natal oogenesis after oocyte depletion, questioning the possible role of OSCs

Histological Observations and Markers

Large ovoid cells were identified in ovarian epithelium, not fitting the characteristics of primordial follicles
Germline markers (MVH/DDX4) and proliferation markers (e.g., BRDU) were used to identify potential stem cells
Meiotic markers (SCP3, Spell 11, DMC1) were detected in adult ovaries
Presence of these markers implies ongoing germ cell production

Additional Experiments

Treatment with Buculfon (a pre-meiotic cell depleter) in mice showed follicle pool depletion and not regeneration
These experiments highlighted that other factors might be involved in follicle regeneration instead of OSCs

Isolation of OSCs

Presumptive OSCs have been isolated from ovaries of adult mice, rats, cows, and humans
The isolation is typically based on the expression of germ cell marker DDX4 or MVH.
Often co-labeled with stem cell markers such as Oct4 (Pou5f1) to confirm their stemness

In Vitro Culture of OSCs

OSCs have been successfully cultured in vitro
These cells express characteristics of germ cells and stem cells, but not mature oocytes

Differentiation and Follicle Formation

OSCs in culture show stem cell characteristics, like high telomerase activity
They can differentiate into large spherical cells (>35 µm) that express oocyte markers
When cultured with granulosa cells, they form follicle-like structures mimicking natural follicle formation

Production of Offspring

A study successfully produced offspring from germline stem cells derived from neonatal ovaries
These stem cells were isolated, labeled with GFP, cultured in vitro, and then transplanted to produce live offspring
These findings support the potential for stem cells to contribute to oocyte generation

Limitations of Existing Studies

The fertility of mice following bone marrow transplantation was not tested regarding the functional implications
Researchers struggled to replicate the findings or isolate OSCs using the initial methods
Some studies failed to detect meiotic markers, contradicting claims of active oogenesis

Single-Cell Analysis of Human Ovarian Cortex

Advanced methodologies (like single-cell RNA sequencing) used to analyze the human ovarian cortex, for the presence of primordial follicles and OSCs
Examination of 21 human patients' ovarian cortex samples

Histology and Tissue Processing

Examined tissue integrity and the presence of primordial follicles in the ovarian cortex
Medullary region of the ovary was dissected, and the cortical tissue was chopped and frozen
A single-cell suspension was created by thawing frozen tissue, and filtering the cells to remove primordial follicles

Absence of Oogonial Stem Cells

The study concluded that no cells with transcriptional profiles compatible with oogonial stem cells were found in adult human ovaries
Cells previously identified with DDX4 were identified as perivascular cells, not stem cells

Challenges with Embryonic Development

Embryo development in cultures from in vitro generated oocytes is often arrested at the cleavage stage
Loss of embryos during early and late gestation
Low success rate of producing full-term offspring from in vitro generated oocytes (3.5% compared to 61.7% in vivo)

Future Directions

Refining in vitro strategies to generate oocytes of higher quality is needed
Creating better culture conditions more closely resembling the human ovary - More research needed to find a viable methodology that mimics natural ovulation and follicle development in human adults

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Description

This quiz explores the in vitro maturation process of mouse oocytes, covering key endpoints, timing, and characteristics of the maturation stages. It also examines recent research findings regarding meiotic markers and the origins of oocyte stem cells. Test your knowledge on this advanced topic in reproductive biology.