- Open Access
The ovarian germinal reserve and apoptosis-related proteins in the infant and adolescent human ovary
© The Author(s). 2019
- Received: 26 October 2018
- Accepted: 19 February 2019
- Published: 11 March 2019
Normal pubertal ovary displays all stages of follicular development and a biased BAX/BCL2 protein ratio in favor of pro-apoptotic BAX protein comparable to the adult ovary. However, adolescents suffering malignant extra-gonadal disease show a limited follicle development after cytotoxic drug treatment and a reduced capacity of in vitro follicle growth. We evaluated the expression of pro- and anti-apoptotic members of the BCL2 gene family, the FAS/FAS-L proteins from the extrinsic apoptosis pathway, the germ-cell-specific marker VASA, the pluripotency marker OCT3/4, and markers of early and late apoptosis in the ovary of pubertal patients with malignant extra-gonadal disease, which received or not pre-surgery chemotherapy, entering a cryopreservation program.
Ovarian biopsies from 12 adolescent girls were screened for follicle count and expression of VASA, OCT3/4, BAX, BCL2, MCL1L and S, cleaved-BID, FAS/FAS-L and CASPASE 3 through immunohistochemistry, western blot and RT-PCR. All stages of folliculogenesis, from primordial to antral follicle, were present in all 12 patients analyzed. VASA and most of the screened apoptosis-related genes showed a pattern of immune-expression comparable to that previously reported. OCT3/4 showed a cytoplasmic localization in the great majority of the primordial follicles; however, in some cases the localization was nuclear. In addition, OCT3/4B showed a significant reduction compared to OCT3/4A. Unexpectedly, BCL2 was detected at all stages of folliculogenesis, associated to the Balbiani’s body in the primordial follicles, regardless of whether patients had or had not received chemotherapy, ruling out the possibility that its expression is a protective response to chemotherapy.
These findings reveal new information on the morphological status of the follicular reserve and the expression of apoptosis-related genes in histologically normal adolescent ovary from patients undergoing extragonadal cancer. The unexpected expression of apoptosis-inhibiting BCL2 protein, both in patients that had or had not received chemotherapy, opens a new avenue for thorough investigations. Moreover, the nuclear localization of OCT3/4 protein in primordial follicle-enclosed oocytes suggests a possible increased activity of ovarian stem cells in response to chemotherapy and/or extragonadal cancer. This new information can be essential for a better managing of in vitro culture of follicles that can be removed by filtration from preserved ovarian tissue, especially in girls that entered a cryopreservation program.
- Human ovary
- Ovarian reserve
- BCL2-family proteins
In women, the establishment of the germinal reserve takes place during fetal life through the interplay between germ cell proliferation and death in the developing ovary. As soon as proliferation begins to increase the 1000–2000 primordial germ cells that reached the developing ovary, to produce around 7 × 106 potential oocytes at mid-fetal life, a counterbalance mechanism of cell death, executed mainly through apoptosis, takes place. The size of the resting primordial follicle reserve is determined by the fourth month of fetal life, as a consequence of cell proliferation/cell death balance, when primordial follicle development begins. Germ cell attrition particularly occurs in the transition from meiotic prophase I to primordial follicle formation through massive apoptosis and continues until the end of gestation, leading the developing ovary to contain just around 1-2 × 106 primordial follicles at birth; a wasteful cellular loss of almost 85% of the potential 7 × 106 oocyte population reached at mid gestation [8, 16]. Death is mainly driven through intrinsic apoptosis mechanisms governed by a concerted expression of BCL2 gene family members  acting in the germ cell proper throughout fetal life [4, 36]. In the adult ovary, germ cell elimination through apoptosis will continue, both in resting and growing follicles, acting mainly in granulosa cells surrounding the oocyte, until the germinal reserve is exhausted and women enter menopause . Although apoptosis occurs under physiological conditions and contributes to maintain cellular homeostasis, an alteration in its regulation may lead to tissue alterations. For example, it was observed that a decrease in apoptosis is present in endometriosis and ovarian cancer [7, 14, 37].
In the last 20 years, several studies analyzing the expression of BCL2-family genes have focused mainly in the fetal and adult ovary, showing that pro-apoptotic members prevail throughout ovarian development, determining the known high rates of oocyte elimination whereas anti-apoptotic genes, like BCL2 and MCL1, show a time-restricted pattern of expression and act as key regulators determining survival and preserving germ cell availability at proliferative time-points [18, 35, 36]. The infant and adolescent ovary deserved less attention probably due to the limited availability of samples. Histological studies performed in the 70s’ have shown that all stages from primordial to early-antral follicles are detected in the infant ovary [27, 28], with the exception of the pre-ovulatory follicle that appears at the onset of puberty , showing that follicle growth is by and large comparable to the adult ovary. However, follicle development is inhibited in children with abdominal tumors or treated with cytotoxic drugs or irradiation [20, 21]. More recently, an analysis of children and adolescent ovary from patients with malignant or chronic illness showed a high proportion of abnormal non-growing follicles with reduced capacity for in vitro development . We have previously analyzed BCL2 and BAX proteins in normal infant and pubertal ovary showing that both proteins behave as in fetal life as far as the oocyte remains in the primordial resting reserve, whereas in follicles entering the growing pool gene expression moves from the germ cell to granulosa cells with an expression pattern comparable to the adult ovary . Nevertheless, no data exist so far regarding the behavior of apoptosis-related proteins in the adolescent ovary from patients suffering malignant extra-gonadal disease whose ovary has been shown to have limited follicle development after cytotoxic drug treatment or irradiation [20, 21] and display a reduced capacity of in vitro follicle development .
The aim of this study was to evaluate the expression of pro- and anti-apoptotic members of the BCL2 gene family, the FAS/FAS-L proteins from the extrinsic apoptosis pathway, the germ-cell-specific marker VASA, the pluripotency marker OCT3/4, and markers of early- and late-apoptosis in the ovary of pubertal patients with malignant extra-gonadal disease, which received or not pre-surgery chemotherapy, entering a cryopreservation program.
Follicle count in cortical ovarian fragments
Follicle count and follicle classification in adolescent human ovarian biopsies from patients with extra-gonadal malignant disease
Number of follicles
Tissue volume (mm3)
Primordial follicles (%)
2 luteum bodies
2 atretics; 2 luteum bodies; 1 albicans body
4 luteum bodies
2 atretics, 1 luteum body, 2 albicans bodies
1 atretic; 1 luteum body; 2 albicans bodies
1 atretic; 2 luteum bodies; 3 albicans body
1 atretic; 1 luteum body; 1 albicans body
1 albicans body
1 luteum body
3 atretics y 1 luteum body
1 atretic y 2 luteum bodies
Expression of VASA and OCT3/4 proteins
Expression of intrinsic apoptosis proteins
Immunolocalization of extrinsic FAS/FAS-L proteins
Caspase-3 detection and TUNEL assay
mRNA expression in adolescent ovary
Our current knowledge about the infant and teen human ovary is still scarce, most likely due to difficulties in obtaining samples from children and adolescents. Besides reports on the general histology performed in the 70s’ [20, 21, 27–29], a few studies have been published more recently analyzing the ovarian histogenesis, follicular composition and immunolocalization of apoptosis- and germline-related proteins [2, 6, 15].
The ovarian reserve and folliculogenesis in the pubertal ovary
Ovarian sections from all 12 patients examined showed an active folliculogenesis and a high density of primordial follicles. The wide variability of primordial follicles, ranging from 50 to 99% of the total follicular pool, among patients (cf. Table 1) may be attributed to the random and the heterogeneous distribution of follicles in the human ovary [16, 32], especially considering that primordial follicles are normally found in clusters . In agreement with previous studies , pubertal ovaries showed all follicular stages, including antral follicles and luteum and albicans bodies as well, confirming the post-menarche beginning of cyclic follicle recruitment. The only pre-pubertal ovary included in this report, a 7 years old patient, showed that histogenesis was still in progress with initial follicle recruitment. We did not observe significant differences in the percentage of primordial follicles between patients that had or had not received chemotherapy before surgery. Although chemotherapy drugs are known to be highly toxic and they can upregulate the PI3K pathway leading to a wave of follicular recruitment and growth that burnout the ovarian reserve , our results agreed with Duncan et al.  in that the adolescent ovary displays follicles irrespective of treatment history.
VASA, OCT3/4 and apoptosis-related proteins in the adolescent ovary
VASA expression was restricted to the cytoplasm of oocytes in primordial and primary follicles in all 12 patients analyzed. In primordial follicles, VASA showed a para-nuclear localization corresponding to Balbiani’s vitelline body, whereas in primary follicles VASA distributed homogeneously in the cytoplasm. No difference with previous observations was detected . It has been proposed that VASA expression follows a stage-specific immune detection pattern that reflects the main changes during fetal ovarian development [4, 5, 12, 34]. In post-natal ovaries, we have found that VASA continues to express after birth until puberty in the resting primordial follicle reserve, associated with the Balbiani’s body; its expression pattern relates mainly to the follicular stage rather than to the stage of development . The association or not of VASA to the Balbiani space may relate to the final fate of the germ cell . In support, the ovary of Lagostomus maximus, a rodent with no germ cell attrition or apoptosis-dependent follicular atresia through constitutive expression of BCL2, shows a low abundance of Balbiani-associated VASA throughout development . The presence of anti-apoptotic BCL2 in Balbiani’s body (see below) in patients with extragonadal cancer might influence the survival of the primordial follicle.
Our analysis of BCL2 family proteins showed that the immunolocalization pattern of pro- and anti-apoptotic members was related to follicular stage. BAX and MCL-1 were detectable throughout folliculogenesis whereas cleaved-BID was restricted to the follicular reserve. The pattern of immunolocalization of BAX was similar to that we previously observed in infant, pubertal and adult ovaries [2, 3]. Unexpectedly, the anti-apoptotic BCL2 protein showed an immunolocalization quite different to that previously reported. This protein was detectable throughout folliculogenesis, including primordial and primary follicles of the germinal reserve. The expression of BCL2 in the primordial reserve both in patients that had or had not received chemotherapy rules out the possibility that BCL2 expression may be linked to a chemo-treatment response, supporting the idea that it is related to the presence of an extragonadal tumor. In previous studies, BCL2 protein was not detectable in dormant primordial follicles and primary follicles, leaving the resting reserve in postnatal ovary from individuals not undergoing a cancer situation. It became detectable in the somatic stratum from secondary follicles, as well as in preantral and antral follicles, co-existing with BAX protein [2, 3]. It is worth to note that BCL2 becomes detectable when proliferative processes occur, i.e. at the moment in which the number of oogonia is actively increasing through mitosis in the fetal ovary or when granulosa cells actively divide during the growing follicular phase in the infant/pubertal ovary [2, 4, 18, 35, 36]. In the light of the expression pattern of OCT3/4 found in these patients, especially the presence of nuclear expression (see above), the unexpected detection of BCL2 in the germinal reserve could be a response to an increased proliferative activity of ovarian stem cells. Alternatively, BCL2 expression could be due to a protective response to the presence of an extragonadal pathological process. Cancer is, per se, a disease that induces oxidative stress, thus generating toxic effects on normal cells, combined with anoxia and deficits in nutrients and antioxidants that cannot compensate the production of free radicals [19, 33]. It is tempting to think that germ cells are responding to survive in the stressing environment. BCL2 detection could be of good prognosis for an eventual future recovery of the gametogenic capacity, considering the protective concurrent expression of OCT3/4.
The FAS/FAS-L system involved in the extrinsic pathway of apoptosis, displayed an immunohistochemical pattern comparable to that found for the apoptosis-inducing BAX protein. This system has deserved little attention, and a few contributions explored its role in the human ovary [3, 13, 24, 30]. It is worth to note that both FAS protein and its ligand, FAS-L, displayed strong immune labeling in the somatic stratum of antral and atretic follicles and the corpus luteum. These observations reinforce the idea that FAS/FAS-L acts mainly in follicular regression and atresia .
The occasional presence of cleaved-caspase 3 in primordial follicles suggests that the process of cell damage would be already at play in some follicles that are going to be recruited to the growing pool and most likely they will enter atresia during or before reaching the antral stage. Only antral follicles, and especially fully-grown follicles, as well as atretic follicles, showed TUNEL and cleaved-caspase 3 positive signals in granulosa and theca cells, indicating an active apoptotic process accompanying follicular atresia.
A long-held tenet supports the idea that mammals’ female are born with a finite and non-renewable endowment of oocytes. Nevertheless, the detection in 2004 by Tilly’s group  of a small cell population in the adult mouse ovary residing in the ovarian surface epithelium and expressing meiotic markers fueled the possibility of female neo-oogenesis after birth. Fourteen years later, although some controversies still persist, the presence of ovarian stem cells that could act to regenerate the germline is a proven fact. The detection, in this report, of anti-apoptotic markers and nuclear OCT3/4 expression in the primordial reserve of adolescent patients undergoing extragonadal cancer adds new information on stem cell activity in response to stress conditions and illuminates an encouraging perspective for the restoration of fertility through the development of reliable culture techniques for in vitro re-establishment of the gametogenic capacity in girls that entered a cryopreservation program.
Recovery of ovarian samples
Diagnosis, age and pre-surgery treatment in oncological patients included in the present study
Acute myeloid leukemia
First cycle of G.A.T.L.A and a second cycle of ifosfamide and carboplatin etoposide, after relapse.
Four cycles of ifosfamide, vincristine, actinomycin, epirubicin,
etoposide and carboplatin.
Inguinal synovial sarcoma
Three cycles of ifosfamide and doxorubicin.
OPPA scheme, two cycles of vincristine, doxorubicin and procarbazine, COOP scheme and two cycles of vincristine, procarbazine and cyclophosphamide.
Cycle 1, 2, 4 and 6 of cyclophosphamide, doxorubicin and vincristine; cycle 3 and 5 of etoposide and cisplatin; 6 cycles of 13-cys-retinoic. Also, she received 3000 cGy radiotherapy.
Cytarabine, idarubicine and etoposide.
Histology, measurement of follicular density and follicle classification
Before fixation in formalin, the fragment of tissue was measured in length, width and thickness for volume calculation. After 24 h fixation, fragments were embedded in paraffin, entirely cut into serial 5 μm sections and every fifth section was stained with hematoxylin-eosin (H&E) for routine histology. The remaining sections were kept for immunohistochemical staining. All ovarian follicles inside the whole tissue fragment were counted and classified according to Gougeon . Only oocyte-containing follicles were included in the counting.
Mounted paraffin sections were dewaxed in xylene, rehydrated in graded alcohols and washed in distilled water. Endogenous peroxidase activity was inhibited by using 0.5% H2O2/methanol (v/v) for 20 min at room temperature. Sections were then blocked for 1 h with 15% normal goat serum or normal rabbit serum in phosphate buffered saline (PBS) and incubated overnight at room temperature with the 1:200 diluted primary antibody: goat polyclonal anti-OCT3/4, goat polyclonal anti-VASA; goat polyclonal anti-BCL2; rabbit polyclonal anti-BAX; goat polyclonal anti-BID; rabbit polyclonal anti-MCL-1; rabbit polyclonal anti-FAS; rabbit polyclonal anti-FASL; rabbit polyclonal anti-PROCASPASE 3 and rabbit polyclonal anti-CLEAVED CASPASE 3. All antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA) except cleaved CASPASE 3 purchased from AbCam (Cambridge, UK). After overnight incubation, slides were rinsed thrice in PBS and incubated for 1 h at room temperature with the appropriate 1:200-diluted biotinylated secondary antibody (Vector Labs, Peterborough, UK). After further washing in PBS, sections were incubated for 30 min with 1:100 diluted streptavidin-peroxidase complexes (ABC kit, Vector Labs, UK). Sections were then washed twice with PBS, and development of peroxidase activity was revealed with 0.05% 3,3′-diaminobenzidine (w/v) and 0.1% H2O2 (v/v) in Tris-HCl. Finally, sections were washed with distilled water and mounted in Canada balsam (Biopack, Buenos Aires, Argentina). Negative controls were processed simultaneously by omitting the primary antibody and/or preincubating the primary antibody with the specific commercial synthetic peptide.
Western blot analysis of VASA, BCL2, BAX, MCL-1 and cleaved-BID proteins
Ovarian fragments preserved at − 80 °C were homogenized in ice-cold lysis buffer containing a protease inhibitor cocktail [0.5 mM phenylmethylsulfonyl fluoride (PMSF); 10 mM leupeptin; 10 mM pepstatin; 10 mM aprotinin], and centrifuged at 1.200 g at 4 °C for 10 min. The supernatant was collected and proteins were quantified using the Bradford Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Total proteins (10 μg for BCL2, BAX and VASA; 20 μg for MCL-1 and cleaved-BID) from tissue extracts were separated by one-dimensional SDS-PAGE (10% for MCL-1 and VASA; 12% for BCL2, BAX and cleaved-BID) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P Transfer membrane, Millipore, Billerica, MA, USA). Membranes were then blocked for 1 h in PBS + 0,1% Tween20 with bovine serum albumin (BSA) or non-fat dry milk depending on the first antibody to be used (3% BSA + 3% non fat dry milk for BAX; 5% non fat dry milk for BCL2, MCL-1 and cleaved-BID). After that, they were incubated 1 h at room temperature with the primary antibody (1400 diluted goat polyclonal anti-BCL2, 1:700 diluted rabbit polyclonal anti-BAX, 1:600 diluted rabbit monoclonal anti-cleaved-BID, 1:700 diluted rabbit polyclonal anti-MCL-1, all antibodies from Santa Cruz Biotechnology, Dallas, USA). Goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was employed at a 1:3000 dilution or rabbit anti-goat IgG horseradish peroxidase-conjugated secondary antibody (Vector Labs, Peterborough, UK) at 1:5000 dilution. The immunoreactive product was visualized using the enhanced chemiluminescence system ECL plus GE (Amersham, Fairfield, Connecticut, USA). Snap-id (Millipore, Billerica, Massachusetts, USA) was employed to analyze VASA expression; a 1:200 dilution for goat polyclonal anti-VASA was used (Santa Cruz Biotecnology, Dallas, TX, USA). Horseradish peroxidase-conjugated second antibody was used at a 1:600 dilution. To confirm equal loading, each membrane was analyzed for β-actin protein expression with Snap-id, demonstrating that the band intensities did not show significant changes between the samples analyzed. Briefly, membranes were incubated with monoclonal anti-β-actin (Sigma, Saint Louis, Missouri, USA) diluted 1:20000. After washing, membranes were incubated with a goat anti-mouse IgG (Bio-Rad, 1:600) conjugated to peroxidase, and then revealed as described above. Stained protein molecular weight markers were used as standards (Fermentas, Vilnius, Lithuania). Densitometry was performed on Scion Image for Windows software (Scion Corporation 2000–2001) and VASA, BCL2, BAX, MCL-1 L, MCL-1S and BID expression was normalized to β-actin.
RNA isolation and real time-PCR
Oligonucleotide primers used for real-time PCR amplification of cDNA obtained after reverse transcription from adolescent human ovary RNA
PRIMERS (5′ → 3′)
F: forward. R: reverse
MCL-1 L (NM_021960.4)
F: AGAAAGTAGTGATACTCAAGGACCAA R: TGACAGAGATTAGCTTCTTCAAAAGT
OCT3/4 A (NM_002701.3)
OCT3/4 B (NM_203289.4)
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling
Detection of DNA fragmentation was performed in formalin-fixed/paraffin-embedded sections by TUNEL technique, using the ‘In Situ Cell Death Detection Kit’ (Roche Diagnostics, Germany) with fluorescein-tagged nucleotides. The procedure followed the manufacturers’ recommendations. Treated sections were examined in an Olympus BX40 microscope by conventional epifluorescence with ultraviolet illumination. In order to confirm negative results, TUNEL-processed sections were incubated with 10 UI/ml DNase II (Sigma Chemical Co., USA) in 50 mM Tris–HCl, pH 7.5, 10 mM Mg2Cl and 1 mg/ml BSA for 10 min at room temperature. After incubation, slides were thoroughly rinsed and treated again according to the TUNEL protocol. Images were captured with an Olympus Camedia C-5060 camera.
Mean and standard error (SEM) were calculated and the InfoStat Software (Version 2012, Grupo InfoStat, Universidad Nacional de Córdoba, Córdoba, Argentina) was used for one-way analysis of variance. A log10 transformation of data was done. Tukey’s test was used when differences between more than two groups were compared. A p-value of less than 0.05 was considered statistically significant.
This research was funded by Fundación Científica Felipe Fiorellino-Universidad Maimónides, Buenos Aires, Argentina and the National Research Council, CONICET, from Argentina.
Availability of data and materials
All data generated or analyzed during the current study are included in this published article.
LZ and RMB collected the samples and provided medical information to patients. MIA and MSA performed lab techniques, analyzed and interpreted results and drafted the manuscript. ADV supervised experiments, analyzed results and edited the final manuscript. All authors read and approved the final manuscript.
All procedures performed in this study were approved by the institutional committees from Universidad Maimónides and from Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina. Informed Consent was obtained from all individual participants, and/or their legal representatives, included in this study. The study followed the ethical standards from the 1964 Helsinki declaration and its later amendments.
Consent for publication
The authors declare that they have no competing interests.
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