Expression of Stat3 in germ cells of developing

and adult mouse ovaries and testes

Katherine Murphy, Luis Carvajal, Leo Medico, Melissa Pepling*

Department of Biology, Syracuse University, 130 College Place, Syracuse, NY 13244, USA

Received 16 November 2004; received in revised form 15 December 2004; accepted 16 December 2004

Abstract

The Signal transducers and activators of transcription (Stat) family of proteins plays diverse roles during differentiation in many tissues.

Stat3 is an essential mammalian gene, critical during embryonic development. In mammals, Stat3 is differentially distributed in the

cytoplasm of mature oocytes and in preimplantation embryos suggesting that Stat3 may be involved in determination of polarity. Here, we

report that Stat3 protein is expressed in the cytoplasm of oocytes from primordial, primary and secondary follicles in the adult ovary and in

developing acrosomes of round spermatids in the adult testis. Stat3 is also expressed in gonocytes, prospermatogonia, oogonia and oocytes of

embryonic and neonatal gonads.

q 2004 Elsevier B.V. All rights reserved.

Keywords: Mouse; Stat3; Gamete biology; Oocyte development; Ovary; Oogenesis; Jak-Stat signaling; Testes; Spermatogenesis; Spermiogenesis

1. Results and discussion

Shortly after their formation, female germ cells undergo

a series of incomplete cell divisions resulting in clusters

called cysts (Pepling and Spradling, 1998). The oogonia

stop dividing and enter meiosis at approximately 13.5 dpc

(days postcoitum), becoming oocytes, eventually arresting

in prophase I. After birth, mouse germ cell cysts break down

into individual oocytes that become surrounded by somatic

pre-granulosa cells to form primordial follicles (Pepling and

Spradling, 2001). During the process of cyst breakdown, a

subset of oocytes in each cyst dies with only a third of the

initial number of oocytes surviving (Pepling and Spradling,

2001). In the adult mouse, cohorts of primordial follicles are

periodically recruited to enter a 3-week growth phase that

ends in meiotic maturation and ovulation (Amleh and Dean,

2002). During this period, the oocyte grows and the

surrounding granulosa cells proliferate. Only some follicles

survive to maturity, the majority undergo atresia during

which the oocyte and surrounding granulosa cells die by

programmed cell death (Hsueh et al., 1994).

1567-133X/$ - see front matter q 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.modgep.2004.12.007

* Corresponding author. Tel.: C1 315 443 4541; fax: C1 315 443 2012.

E-mail address: mepeplin@syr.edu (M. Pepling).

In the male, primordial germ cells aggregate with Sertoli

cells to form seminiferous cords enclosed by peritubular

cells (Mackay, 2000; Orth et al., 2000). Male germ cells

divide until approximately 13.5 dpc when they exit the cell

cycle and remain quiescent as gonocytes until a few days

after birth (Monk and McLaren, 1981). The gonocytes are

initially in a central position within the cords but in the

neonate relocate to the basement membrane at the periphery

of the tubules, become prospermatogonia and eventually

develop into spermatogonial stem cells that produce

spermatozoa (Brinster, 2002; Mackay, 2000; Orth et al.,

2000). Differentiation of spermatogonia into spermatozoa

takes about 5 weeks and begins when the germ cells enter

meiosis and become spermatocytes. The result of meiosis is

the formation of haploid spermatids that undergo spermio-

genesis to form mature spermatozoa. During spermiogen-

esis, the acrosome, a secretory organelle important for

sperm-egg penetration, begins to form (Ramalho-Santos

et al., 2002).

The Signal transducers and activators of transcription

(Stat) family of proteins is important during differentiation

in many tissues (for reviews see (Ihle, 2001; Kisseleva et al.,

2002)). Membrane receptors are activated by ligand

binding, and in turn, activate Jak family protein kinases,

Gene Expression Patterns 5 (2005) 475–482

www.elsevier.com/locate/modgep

K. Murphy et al. / Gene Expression Patterns 5 (2005) 475–482476

which recruit and phosphorylate Stats. Phosphorylated Stat

proteins homodimerize and translocate to the nucleus where

they regulate transcription of target genes. This pathway can

be activated by a large number of cytokines and growth

factors in vertebrates (Hou et al., 2002). In mammals, there

are seven known Stat proteins (Kisseleva et al., 2002).

Fig. 1. Stat3 protein expression in adult mouse ovaries and testes. (A–C) low p

propidium iodide (B) and overlay (C). Arrows in A and C indicate examples of l

primordial and primary follicles (D), a secondary follicle (E), and a preantral fo

antibody (G), propidium iodide (H) and overlay (I) showing labeling of developing

adult testis showing Stat3 antibody staining in (J) and lack of staining of spermatog

(red). Scale barZ50 mm A–C,E,F, Z20 mm, D,G–I and Z10 mm, J,K.

In addition, the Stat family proteins are well conserved and

homologous proteins have been found in C. elegans,

Dictyostelium, Drosophila and zebrafish (Hou et al., 2002).

Stat3 is essential during mammalian development

because mice with a targeted disruption of Stat3 die as

embryos (Takeda et al., 1997). Studies of cultured cells and

ower confocal section of an adult ovary labeled with Stat3 antibody (A),

abeled oocytes. High power view of Stat3 protein expression in oocytes of

llicle (F). (G–K) Confocal section of adult mouse testis labeled with Stat3

acrosomes in round spermatids. High power view of a confocal section from

onia, spermatocytes and Sertoli cells in (K). Stat3 (green), propidium iodide

Fig. 2. Stat3 mRNA and protein expression in mouse ovaries and testes. (A)

RT-PCR analysis. RNA was isolated from 13.5 dpc, PND 1, PND 4 and

PND 42 ovaries and testes and Stat3 transcripts were monitored by RT-

PCR. GAPDH was also monitored as a loading control. (B) Western

blotting analysis. Mouse tissue extracts from PND 1, PND 4 and PND 42

ovaries and testes were probed with the Stat3 antibody. A band of

approximately 92 kDa corresponding to the molecular mass of Stat3a was

detected in PND 1 and 4 ovaries while an 84 kDa band corresponding to

Stat3b was detected in PND 1 and 4 testes as well as PND 42 ovaries and

testes. Blots were reprobed with GAPDH (38 kDa) as a loading control.

K. Murphy et al. / Gene Expression Patterns 5 (2005) 475–482 477

of tissue specific knockouts suggest that Stat3 can function

in maintaining pluripotency or in promoting differentiation

depending on the tissue or cell line studied (Levy and Lee,

2002). In mouse embryonic stem cells, activated Stat3

suppresses differentiation (Matsuda et al., 1999; Niwa et al.,

1998; Raz et al., 1999), while in monocytes, Stat3 is

involved in terminal differentiation and growth arrest

(Heinrich et al., 1998).

Stats are also important in the process of gametogenesis.

In the Drosophila testis, germline stem cells are maintained

by the Jak-Stat pathway (Kiger et al., 2001; Tulina and

Matunis, 2001). In the Drosophila ovary, Jak-Stat signaling

participates in differentiation of somatic support cells (Xi

et al., 2003). In mammals, Stat3 is differentially distributed

in the cytoplasm of mature oocytes as well as in

preimplantation embryos (Antczak and Van Blerkom,

1997) suggesting that Stat3 may be involved in the

determination of polarity. However, because the Stat3

knockout is embryonic lethal its role in oogenesis has not

yet been determined.

In an effort to identify molecules involved in determining

the polarity in early mouse ovarian germ line cysts, we

asked whether Stat3 is expressed in embryonic and neonatal

gonads. We find Stat3 protein present in the cytoplasm of

oocytes from primordial, primary and secondary follicles in

the adult mouse ovary and in the developing acrosome in the

adult testis. In embryonic and neonatal gonads, we find

Stat3 expressed in oogonia and oocytes in the ovary as well

as in gonocytes and prospermatogonia in the testes.

1.1. Stat3 expression in adult ovaries and testes

Although Stat3 protein is present in isolated fully grown

oocytes (Antczak and Van Blerkom, 1997), it was not

known if Stat3 protein was expressed in oocytes from

developing follicles or in other cells of the adult mouse

ovary. We examined the expression of Stat3 protein in

postnatal day (PND) 42 mouse ovaries and detected Stat3

in oocytes but not in granulosa or other somatic cells

(Fig. 1A–C). Specifically, Stat3 is detected in the cytoplasm

of oocytes from primordial, primary and secondary follicles

(Fig. 1D,E) but not in preantral or antral follicles (Fig. 1F).

In PND 42 testes, Stat3 is located in the developing

acrosomes of round spermatids (Fig. 1G–J) but not in Sertoli

cells, spermatogonia or spermatocytes (Fig. 1K).

1.2. RT-PCR and Western Blot Hybridization

To determine if Stat3 was expressed in developing

ovaries and testes, RT-PCR and Western blotting techniques

were used. Stat3 mRNA was detected by RT-PCR in both

ovaries and testes at 13.5 dpc, PND 1, PND 4 and PND 42

(Fig. 2A). Stat3 protein was detected by Western blotting in

both ovaries and testes at PND 1, PND 4 and PND 42

(Fig. 2B). Two different isoforms of Stat3 are produced by

alternative splicing (Schaefer et al., 1995). The longer form

is Stat3a with a molecular weight of 92 kd. Stat3b is a

shorter form in which 55 C-terminal amino acids are

replaced by seven new amino acids. The relative amounts of

each isoform varies depending on cell type, ligand exposure

and maturation stage (Schaefer et al., 1997). The Stat3bisoform is thought to function as a dominant negative

inhibitor of Stat3a (Yoo et al., 2002). We found that in adult

ovaries and testes and in developing testes the predominant

form was Stat3b while in developing ovaries the predomi-

nant form present was Stat3a (Fig. 2B).

1.3. Expression of Stat3 in developing ovaries and testes

To determine what cell types express Stat3 protein in

developing embryonic and neonatal gonads, whole mount

immunocytochemistry was used. Stat3 is not detected at

12.5 dpc (Figs. 3A–C and 4A–C) but begins to be expressed

at 13.5 dpc in developing ovaries and testes (Figs. 3D–F and

4D–F). At 13.5 dpc, there is a low level of Stat3 in all cells of

both ovaries and testes but expression is stronger in the germ

cells. In the ovary, Stat3 becomes progressively stronger in

the cytoplasm of oocytes while expression in somatic cells

becomes weaker (Fig. 3G–I) until by PND4 Stat3 is not

detected in the somatic cells (Fig. 3J–L). A similar pattern is

observed in the testes, however the labeling is not as strong in

the cytoplasm of the spermatogonia and weak labeling of the

Sertoli cell cytoplasm is also detected (Fig. 4G–L).

Fig. 3. Expression pattern of Stat3 protein in developing ovaries. Immunofluorescence of single confocal ovary sections at 12.5 dpc (A–C), 13.5 dpc (D–F),

PND 1 (G–I), and PND 4 (J–L) showing no labeling with Stat3 at 12.5 dpc (A) and oogonia and oocytes labeled with Stat3 antibody (D,G,J, green), nuclei

labeled with propidium iodide (B,E,H,K, red) and overlay (C,F,I,L). Arrow in (J–L) shows an example of Stat3 cytoplasmic labeling in an oocyte and

arrowhead indicates a granulosa cell that is not labeled with Stat3. Scale barZ20 mm.

K. Murphy et al. / Gene Expression Patterns 5 (2005) 475–482478

1.4. Colocalization with germ cell markers

To confirm that Stat3 was specifically labeling germ

cells in the developing gonads, immunocytochemistry

using the germ cell specific antibodies, EMA-1, Vasa or

Germ Cell Nuclear Antigen (GCNA) was employed in

combination with the Stat3 antibody. EMA-1 antibody

labels germ cells from 8.5 to 13.5 dpc and recognizes a

carbohydrate on the surface of these cells (Hahnel and

Eddy, 1986). In 13.5 dpc ovaries, EMA-1 and Stat3 were

detected in the same cells (Fig. 5A–D). GCNA labels

male and female germ cells from the time they arrive at

Fig. 4. Expression pattern of Stat3 protein in developing testes. Immunofluorescence of single confocal testis sections at 12.5 dpc (A–C), 13.5 dpc (D–F),

PND 1 (G–I), and PND 4 (J–L) showing no labeling with Stat3 at 12.5 dpc (A) and prospermatogonia labeled with Stat3 antibody (D,G,J, green), nuclei labeled

with propidium iodide (B,E,H,K, red) and overlay (C,F,I,L). White arrow in G–I shows an example of Stat3 cytoplasmic labeling in a prospermatogonium,

white arrowhead indicates a Sertoli cell nucleus and gray arrow shows Sertoli cell cytoplasm weakly labeled with Stat3. Scale barZ20 mm.

K. Murphy et al. / Gene Expression Patterns 5 (2005) 475–482 479

the genital ridge until they enter the diplotene stage of

meiosis I (Enders and May, 1994). PND 1 ovaries and

testes were exposed to both Stat3 and GCNA antibodies

and Stat3 and GCNA were detected in the same cells

(Fig. 5E–L). PND 1 ovaries were also exposed to

antibodies for Stat3 and Vasa which is expressed in the

cytoplasm of male and female germ cells beginning at

11.5 dpc (Tanaka et al., 2000) and expression of these two

proteins overlapped (Fig. 5M–P).

We have identified Stat3 as a germ cell marker in mice

and as only a few germ cell markers are known, Stat3 is a

useful tool for germ cell studies. The role of Stat3 in the

cytoplasm of germ cells is unknown. When Stat3 is

activated, it moves from the cytoplasm to the nucleus.

Fig. 5. Colocalization of Stat3 and germ cell marker expression in developing ovaries and testes. Immunofluorescence of single confocal sections. (A–D)

13.5 dpc ovaries labeled with EMA (green), Stat3 (red) and Toto-3 (blue). (E–L) PND 1 ovaries (E–H) and testes (I–L) labeled with GCNA (green), Stat3 (red)

and Toto-3 (blue). (M-P) PND 1 ovaries labeled with Stat3 (green), vasa (red) and Toto-3 (blue). Scale barZ20 mm.

K. Murphy et al. / Gene Expression Patterns 5 (2005) 475–482480

We did not observe strong nuclear staining at any of the

stages examined. Therefore, Stat3 may not be active in germ

cells, or only a small fraction of the protein may be active.

In oocytes, it may be present so that it is available for use in

early embryogenesis. Characterization of Stat3 function in

the ovary and the testes awaits the generation of gonad

specific knockouts.

2. Materials and methods

2.1. Mice

Ovaries and testes from embryos, pups and adults were

obtained from wildtype CD-1 mice (Charles River Labs).

The presence of a vaginal plug the morning after mating was

K. Murphy et al. / Gene Expression Patterns 5 (2005) 475–482 481

designated 0.5 dpc. Birth occurred at 19.5 dpc and was

designated PND 1. All animal experimentation was

reviewed and approved by the Syracuse University

Institutional Animal Care and Use Committee.

2.2. RT-PCR

Total RNA was prepared from embryonic and neonatal

gonads using Trizol (Life Technologies, Inc.) 20 gonads

were used at each timepoint and RNA was resuspended in

25 ml PCR grade water. First strand cDNA was synthesized

from 1 ml total RNA using Superscript II reverse transcrip-

tase and 3 0 RACE primer (Invitrogen). The cDNA was then

amplified with Stat3 forward and reverse primers: Stat3

forward: 5 0 TGC CCA TGG CTA CCT GTT 3 0, Stat3

reverse: 5 0 GAA CCT CCT GGG CTT AGT CC 3 0. As a

positive control in RT-PCR experiments, the constitutively

expressed mRNA for Glyceraldehyde-3-phosphate dehy-

drogenase (GAPDH) was RT-PCR amplified in tandem with

experimental samples using the following primers: 5 0YGC

CTG CTT CAC CAC CTT C 3 0, 5 0 TGC MTC CTG CAC

CAC CAA CT 3 0 where YZT or C and MZA or C (Sigma).

2.3. Western blot hybridization

Gonads were homogenized in Sample Buffer (2%

SDS, 10% glycerol, 25 mM Tris pH 6.8, 0.00005%

Bromophenol Blue, 0.025% mercaptoethanol) plus mini

complete protease inhibitor (Sigma). PND 1 and 4

ovaries were homogenized in 10 ml sample buffer/ovary

and PND 1 and 4 testes in 20 ml/testes. Adult ovaries

were homogenized in 200 ml/ovary and adult testes in

800 ml/testis. For each sample, 20 ml solubilized protein

extract was mixed with 1/10 volume mercaptoethanol,

heated to 95 8C for 3 min, separated on 10% SDS-

polyacrylamide gels and electroblotted onto Immobilon P

membranes (Millipore). The blots were incubated with a

blocking solution containing 5% non-fat milk powder in

PBST (PBS/0.05% Tween 20) overnight at 4 8C and

incubated with Stat3 antibody in blocking solution for

1 h at room temperature. Following three washes in

blocking solution, membranes were incubated with

horseradish peroxidase-conjugated secondary IgG

(1:10,000) in blocking solution at room temperature for

1 h, washed in PBST three times and signal visualized

using the Supersignal kit (Pierce) on films. Blots were

reprobed for GAPDH as a loading control.

2.4. Antibodies

The Stat3 (C20) antibody (Santa Cruz Biotechnology)

was used at a dilution of 1:500 in immunocytochemistry

and 1:1000 in Western blotting. GAPDH antibody

(EnCor Biotechnology) was used at 1:2000. The EMA

antibody was obtained from the developmental studies

hybridoma bank and used at a dilution of 1:1, the GCNA

antibody used at 1:1 was a gift of George Enders and the

Vasa antibody used at 1:500 was a gift of Toshiaki Noce.

For double labeling with Stat3 and Vasa, Stat3 antibody

(Upstate) was directly labeled with Alexa Fluor 488

using the Alexa Fluor 488 Monoclonal Antibody

Labeling Kit (Molecular Probes).

2.5. Immunohistochemistry

Whole ovaries and testes from embryos, neonate and

adults of the wildtype CD-1 strain were harvested, fixed in

5% EM grade paraformaldehyde (Ted Pella, Inc.) and

stained as previously described (Pepling and Spradling,

1998) except that neonatal and adult ovaries and testes were

fixed overnight at 4 8C. After adult ovaries and testes were

fixed, they were sliced into small pieces before incubation

with antibody. Propidium iodide or Toto-3 (Molecular

Probes) was used to label nuclei. Samples were imaged on a

Zeiss Pascal Confocal microscope.

Acknowledgements

We thank Gerda Breitwieser, Scott Erdman, Jodi Flaws,

Eleanor Maine and John Young for helpful discussions and

Toshiaki Noce and George Enders for their generous gifts of

antibodies. EMA-1 antibody was obtained from the

Developmental Studies Hybridoma bank developed under

the auspices of the NICHD and maintained by the

University of Iowa, Department of Biological Sciences,

Iowa City, IA 52242.

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