PUMA regulates germ cell loss and primordial follicle endowment in mice

The number of primordial follicles initially established within the ovary is influenced by the extent of germ cell death during foetal ovarian development, but the mechanisms that mediate this death have not been fully uncovered. In this study, we identified BBC3 (PUMA) (p53 upregulated modulator of apoptosis, also known as BCL2-binding component 3), a pro-apoptotic BH3-only protein belonging to the BCL2 family, as a critical determinant of the number of germ cells during ovarian development. Targeted disruption of the Bbc3 gene revealed a significant increase in the number of germ cells as early as embryonic day 13.5. The number of germ cells remained elevated in Bbc3−/− female mice compared with WT female mice throughout the remainder of embryonic and early postnatal life, resulting in a 1.9-fold increase in the number of primordial follicles in the ovary on postnatal day 10. The increase in the number of germ cells observed in the ovaries of Bbc3−/− mice could not be attributed to the altered proliferative activity of germ cells within the ovaries. Furthermore, BBC3 was found to be not required for the massive germ cell loss that occurs during germ cell nest breakdown. Our data indicate that BBC3 is a critical regulator of germ cell death that acts during the migratory phase of oogenesis or very soon after the arrival of germ cells in the gonad and that BBC3-mediated cell death limits the number of primordial follicles established in the initial ovarian reserve.

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127. PUMA MEDIATES GERM CELL DEATH DURING OVARIAN DEVELOPMENT AND DETERMINES INITIAL PRIMORDIAL FOLLICLE NUMBER IN MICE

Reproduction Fertility and Development ◽

10.1071/srb10abs127 ◽

2010 ◽

Vol 22 (9) ◽

pp. 45

Author(s):

F. Morgan ◽

K. J. Hutt ◽

C. L. Scott ◽

M. Cook ◽

A. Strasser ◽

...

Keyword(s):

New York ◽

Cell Death ◽

Germ Cell ◽

Germ Cells ◽

Ovarian Reserve ◽

Ovarian Development ◽

Primordial Follicle ◽

Primordial Follicles ◽

Apoptotic Protein ◽

Programmed Death

The proteins that control the number of primordial follicles initially established within the ovary are largely unknown. Here we investigated the hypothesis that PUMA, a pro-apoptotic protein belonging to the Bcl-2 family, regulates germ cell death during ovarian development and thereby determines the number of primordial follicles that make up the ovarian reserve. Ovaries were obtained from embryonic day 17.5 (E17.5) and post-natal day 10 (PN10) wild-type (wt) and puma–/– mice and subjected to morphological, molecular and stereological characterisation (n = 3-6 mice/genotype/age). At E17.5, ovaries were densely populated with germ cells and early meiotic oocytes. Immunostaining for MVH and PCNA confirmed the identity of germ cells and proliferating germ cells, respectively. Pyknotic nuclei and TUNEL positive germ cells were rarely detected, suggesting that cell death was uncommon at this age. At PN10, primordial follicle assembly was complete for both genotypes, as confirmed morphologically and by immunostaining for oocyte markers GCNA and MSY2. The number of germ cells in E17.5 wt and puma–/– ovaries was comparable (p=0.81, See Table 1). However, PN10 puma–/– ovaries contained significantly more primordial follicles than wt ovaries (P < 0.001, See Table 1), revealing an over-endowment of primordial follicles in the absence of PUMA. These data show that PUMA regulates the developmentally programmed death of germ cells between E17.5 and PN10 in the mouse and thereby determines the number of primordial follicles that make up the initial ovarian reserve. This work was supported by the NHMRC (Program Grants #494802 and #257502, Fellowships JKF (#441101), KJH (#494836), CLS (#406675), AS (#461299)); the Leukemia and Lymphoma Society (New York; SCOR grant#7015), the National Cancer Institute (NIH, US; CA80188 and CA43540) and Victorian Government Infrastructure Funds.

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Suppression of Notch Signaling in the Neonatal Mouse Ovary Decreases Primordial Follicle Formation

Endocrinology ◽

10.1210/en.2008-0213 ◽

2009 ◽

Vol 150 (2) ◽

pp. 1014-1024 ◽

Cited By ~ 102

Author(s):

Daniel J. Trombly ◽

Teresa K. Woodruff ◽

Kelly E. Mayo

Keyword(s):

Germ Cell ◽

Notch Signaling ◽

Cell Fate ◽

Germ Cells ◽

Ex Vivo ◽

Primordial Follicle ◽

Neonatal Mouse ◽

Postnatal Life ◽

Mouse Ovary ◽

Primordial Follicles

Notch signaling directs cell fate during embryogenesis by influencing cell proliferation, differentiation, and apoptosis. Notch genes are expressed in the adult mouse ovary, and roles for Notch in regulating folliculogenesis are beginning to emerge from mouse genetic models. We investigated how Notch signaling might influence the formation of primordial follicles. Follicle assembly takes place when germ cell syncytia within the ovary break down and germ cells are encapsulated by pregranulosa cells. In the mouse, this occurs during the first 4–5 d of postnatal life. The expression of Notch family genes in the neonatal mouse ovary was determined through RT-PCR measurements. Jagged1, Notch2, and Hes1 transcripts were the most abundantly expressed ligand, receptor, and target gene, respectively. Jagged1 and Hey2 mRNAs were up-regulated over the period of follicle formation. Localization studies demonstrated that JAGGED1 is expressed in germ cells prior to follicle assembly and in the oocytes of primordial follicles. Pregranulosa cells that surround germ cell nests express HES1. In addition, pregranulosa cells of primordial follicles expressed NOTCH2 and Hey2 mRNA. We used an ex vivo ovary culture system to assess the requirement for Notch signaling during early follicle development. Newborn ovaries cultured in the presence of γ-secretase inhibitors, compounds that attenuate Notch signaling, had a marked reduction in primordial follicles compared with vehicle-treated ovaries, and there was a corresponding increase in germ cells that remained within nests. These data support a functional role for Notch signaling in regulating primordial follicle formation. Gamma secretase inhibitor treatment suppresses germ cell nest breakdown in the neonatal mouse ovary, supporting a role for Notch signaling in promoting primordial follicle formation.

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BCL2-modifying factor promotes germ cell loss during murine oogenesis

Reproduction ◽

10.1530/rep-15-0561 ◽

2016 ◽

Vol 151 (5) ◽

pp. 553-562 ◽

Cited By ~ 4

Author(s):

Kavitha Vaithiyanathan ◽

Seng H Liew ◽

Nadeen Zerafa ◽

Thilini Gamage ◽

Michele Cook ◽

...

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Germ Line ◽

Cell Loss ◽

Regulatory Proteins ◽

Primordial Follicles ◽

Large Numbers ◽

Female Germ Line ◽

Low Levels

Abstract Apoptosis plays a prominent role during ovarian development by eliminating large numbers of germ cells from the female germ line. However, the precise mechanisms and regulatory proteins involved in germ cell death are yet to be determined. In this study, we characterised the role of the pro-apoptotic BH3-only protein, BCL2-modifying factor (BMF), in germ cell apoptosis in embryonic and neonatal mouse ovaries. BMF protein was immunohistochemically localised to germ cells at embryonic days 15.5 (E15.5) and E17.5 and postnatal day 1 (PN1), coincident with entry into the meiotic prophase, but was undetectable at E13.5, and only present at low levels at PN3 and PN5. Consistent with this expression pattern, loss of BMF in female mice was associated with a decrease in apoptosis at E15.5 and E17.5. Furthermore, increased numbers of germ cells were found in ovaries from Bmf−/− mice compared with WT animals at E15.5 and PN1. However, germ cell numbers were comparable between Bmf−/− and WT ovaries at PN3, PN5 and PN10. Collectively, these data indicate that BMF mediates foetal oocyte loss and its action limits the maximal number of germ cells attained in the developing ovary, but does not influence the number of primordial follicles initially established in ovarian reserve.

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Spontaneous Germ Cell Apoptosis in Humans: Evidence for Ethnic Differences in the Susceptibility of Germ Cells to Programmed Cell Death

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.83.1.4485 ◽

1998 ◽

Vol 83 (1) ◽

pp. 152-156 ◽

Cited By ~ 72

Author(s):

Amiya P. Sinha Hikim ◽

Christina Wang ◽

Yanhe Lue ◽

Larry Johnson ◽

X.-H Wang ◽

...

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Germ Cell ◽

Ethnic Differences ◽

Germ Cells ◽

Cell Apoptosis ◽

Germ Cell Apoptosis

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Genetic control of programmed cell death in the Caenorhabditis elegans hermaphrodite germline

Development ◽

10.1242/dev.126.5.1011 ◽

1999 ◽

Vol 126 (5) ◽

pp. 1011-1022 ◽

Cited By ~ 8

Author(s):

T.L. Gumienny ◽

E. Lambie ◽

E. Hartwieg ◽

H.R. Horvitz ◽

M.O. Hengartner

Keyword(s):

Cell Death ◽

Caenorhabditis Elegans ◽

Germ Cell ◽

Somatic Cell ◽

Cell Fate ◽

Germ Cells ◽

Mapk Pathway ◽

Apoptotic Cell ◽

C Elegans ◽

Pathway Activation

Development of the nematode Caenorhabditis elegans is highly reproducible and the fate of every somatic cell has been reported. We describe here a previously uncharacterized cell fate in C. elegans: we show that germ cells, which in hermaphrodites can differentiate into sperm and oocytes, also undergo apoptotic cell death. In adult hermaphrodites, over 300 germ cells die, using the same apoptotic execution machinery (ced-3, ced-4 and ced-9) as the previously described 131 somatic cell deaths. However, this machinery is activated by a distinct pathway, as loss of egl-1 function, which inhibits somatic cell death, does not affect germ cell apoptosis. Germ cell death requires ras/MAPK pathway activation and is used to maintain germline homeostasis. We suggest that apoptosis eliminates excess germ cells that acted as nurse cells to provide cytoplasmic components to maturing oocytes.

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Germ cell connectivity enhances cell death in response to DNA damage in the Drosophila testis

eLife ◽

10.7554/elife.27960 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 16

Author(s):

Kevin L Lu ◽

Yukiko M Yamashita

Keyword(s):

Dna Damage ◽

Cell Death ◽

Germ Cell ◽

Germ Cells ◽

High Sensitivity ◽

Next Generation ◽

Drosophila Testis

Two broadly known characteristics of germ cells in many organisms are their development as a ‘cyst’ of interconnected cells and their high sensitivity to DNA damage. Here we provide evidence that in the Drosophila testis, connectivity serves as a mechanism that confers to spermatogonia a high sensitivity to DNA damage. We show that all spermatogonia within a cyst die synchronously even when only a subset of them exhibit detectable DNA damage. Mutants of the fusome, an organelle that is known to facilitate intracyst communication, compromise synchronous spermatogonial death and reduces overall germ cell death. Our data indicate that a death-promoting signal is shared within the cyst, leading to death of the entire cyst. Taken together, we propose that intercellular connectivity supported by the fusome uniquely increases the sensitivity of the germline to DNA damage, thereby protecting the integrity of gamete genomes that are passed on to the next generation.

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Germ Cell Loss Induced by 12C6+ Ion Irradiation in Young Female Mice

Journal of Radiation Research ◽

10.1269/jrr.47.131 ◽

2006 ◽

Vol 47 (2) ◽

pp. 131-134 ◽

Cited By ~ 7

Author(s):

Hong ZHANG ◽

Xu ZHANG ◽

Zhigang YUAN ◽

Xiaoda LI ◽

Wenjian LI ◽

...

Keyword(s):

Germ Cell ◽

Ion Irradiation ◽

Cell Loss ◽

Young Female ◽

Female Mice

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Follistatin Regulates Germ Cell Nest Breakdown and Primordial Follicle Formation

Endocrinology ◽

10.1210/en.2010-0950 ◽

2010 ◽

Vol 152 (2) ◽

pp. 697-706 ◽

Cited By ~ 35

Author(s):

Fuminori Kimura ◽

Lara M. Bonomi ◽

Alan L. Schneyer

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Ovarian Function ◽

Primordial Follicle ◽

Biochemical Properties ◽

The Body ◽

Protein Isoforms ◽

Primordial Follicles ◽

Reduced Rate ◽

Regulatory Functions

Abstract Follistatin (FST) is an antagonist of activin and related TGFβ superfamily members that has important reproductive actions as well as critical regulatory functions in other tissues and systems. FST is produced as three protein isoforms that differ in their biochemical properties and in their localization within the body. We created FST288-only mice that only express the short FST288 isoform and previously reported that females are subfertile, but have an excess of primordial follicles on postnatal day (PND) 8.5 that undergo accelerated demise in adults. We have now examined germ cell nest breakdown and primordial follicle formation in the critical PND 0.5–8.5 period to test the hypothesis that the excess primordial follicles derive from increased proliferation and decreased apoptosis during germ cell nest breakdown. Using double immunofluorescence microscopy we found that there is virtually no germ cell proliferation after birth in wild-type or FST288-only females. However, the entire process of germ cell nest breakdown was extended in time (through at least PND 8.5) and apoptosis was significantly reduced in FST288-only females. In addition, FST288-only females are born with more germ cells within the nests. Thus, the excess primordial follicles in FST288-only mice derive from a greater number of germ cells at birth as well as a reduced rate of apoptosis during nest breakdown. These results also demonstrate that FST is critical for normal regulation of germ cell nest breakdown and that loss of the FST303 and/or FST315 isoforms leads to excess primordial follicles with accelerated demise, resulting in premature cessation of ovarian function.

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Overexpression of peroxisomal testis-specific 1 protein induces germ cell apoptosis and leads to infertility in male mice

Molecular Biology of the Cell ◽

10.1091/mbc.e09-12-0993 ◽

2011 ◽

Vol 22 (10) ◽

pp. 1766-1779 ◽

Cited By ~ 16

Author(s):

Karina Kaczmarek ◽

Maja Studencka ◽

Andreas Meinhardt ◽

Krzysztof Wieczerzak ◽

Sven Thoms ◽

...

Keyword(s):

Cell Death ◽

Germ Cell ◽

Germ Cells ◽

Specific Gene ◽

Male Mice ◽

Terminal Part ◽

Male Germ Cells ◽

Germ Cell Apoptosis ◽

Induced Apoptosis

Peroxisomal testis-specific 1 gene (Pxt1) is the only male germ cell–specific gene that encodes a peroxisomal protein known to date. To elucidate the role of Pxt1 in spermatogenesis, we generated transgenic mice expressing a c-MYC-PXT1 fusion protein under the control of the PGK2 promoter. Overexpression of Pxt1 resulted in induction of male germ cells’ apoptosis mainly in primary spermatocytes, finally leading to male infertility. This prompted us to analyze the proapoptotic character of mouse PXT1, which harbors a BH3-like domain in the N-terminal part. In different cell lines, the overexpression of PXT1 also resulted in a dramatic increase of apoptosis, whereas the deletion of the BH3-like domain significantly reduced cell death events, thereby confirming that the domain is functional and essential for the proapoptotic activity of PXT1. Moreover, we demonstrated that PXT1 interacts with apoptosis regulator BAT3, which, if overexpressed, can protect cells from the PXT1-induced apoptosis. The PXT1-BAT3 association leads to PXT1 relocation from the cytoplasm to the nucleus. In summary, we demonstrated that PXT1 induces apoptosis via the BH3-like domain and that this process is inhibited by BAT3.

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