Pubertal Ovaries Research Articles

Single-cell multi-omics profiling reveals key regulatory mechanisms that poise germinal vesicle oocytes for maturation in pigs.

The molecular mechanisms controlling the transition from meiotic arrest to meiotic resumption in mammalian oocytes have not been fully elucidated. Single-cell omics technology provides a new opportunity to decipher the early molecular events of oocyte growth in mammals. Here we focused on analyzing oocytes that were collected from antral follicles in different diameters of porcine pubertal ovaries, and used single-cell M&T-seq technology to analyze the nuclear DNA methylome and cytoplasmic transcriptome in parallel for 62 oocytes. 10× Genomics single-cell transcriptomic analyses were also performed to explore the bi-directional cell-cell communications within antral follicles. A new pipeline, methyConcerto, was developed to specifically and comprehensively characterize the methylation profile and allele-specific methylation events for a single-cell methylome. We characterized the gene expressions and DNA methylations of individual oocyte in porcine antral follicle, and both active and inactive gene's bodies displayed high methylation levels, thereby enabled defining two distinct types of oocytes. Although the methylation levels of Type II were higher than that of Type I, Type II contained nearly two times more of cytoplasmic transcripts than Type I. Moreover, the imprinting methylation patterns of Type II were more dramatically divergent than Type I, and the gene expressions and DNA methylations of Type II were more similar with that of MII oocytes. The crosstalk between granulosa cells and Type II oocytes was active, and these observations revealed that Type II was more poised for maturation. We further confirmed Insulin Receptor Substrate-1 in insulin signaling pathway is a key regulator on maturation by in vitro maturation experiments. Our study provides new insights into the regulatory mechanisms between meiotic arrest and meiotic resumption in mammalian oocytes. We also provide a new analytical package for future single-cell methylomics study.

P-417 Biovular follicles are commonly found in prepubertal but not adult ovaries

Abstract Study question Is the morphology of ovarian tissue from paediatric and adolescent girls similar to that from adults? Summary answer The presence of biovular follicles was more frequently observed in paediatric ovary than in the adult ovary. What is known already At birth there are approximately 1 million ovarian follicles that reduce to around 380,000 by puberty. Limited information is available on the morphology of paediatric and adolescent ovary but it is thought that, before the age of 6 years, the prepubertal ovary contains around 20% abnormal primordial follicles - more than in the adult ovary. Polyovular follicles are common in some animal species but rarely observed in the human adult ovary. They are thought to contribute to dizygotic twinning but the low frequency suggests they are not the sole origin. Study design, size, duration Haematoxylin and eosin stained sections of ovarian tissue were examined by 2 operators blinded to patient age. Follicles were classified according to modified Gougeon criteria. The proportions were compared with Fisher’s exact test and the frequency by one- way ANOVA. Participants/materials, setting, methods Ovarian cortex tissue was collected from a total of 71 children and adolescents and 85 adults undergoing fertility preservation. Those who had had previous chemotherapy and or pelvic irradiation were excluded. The population was divided into 30 children (<8 years, range 1 - 7.6 years), 25 peripubertal (8 - 14.1 years), 16 with confirmed pubertal status (13.7 - 16.6 years) and compared to 85 adults (18-36 years). Main results and the role of chance Biovular follicles were observed in 36.7% (11/30) of the ovarian tissue from children <8 years with, where present, an average frequency of 1/10.6 x103 follicles, in 28% (7/25) of the peripubertal girls with an average frequency of 1/11.9x103 follicles, and 18.7% (3/16) of the post pubertal girls with an average frequency of 1/7.7x103 follicles. In contrast, biovular follicles were rarely seen in the adult ovarian tissue examined (3.5%, 3/85) with an average frequency of 1/25x103 follicles. The observed frequency in all of the younger samples was significantly different to the adult (p < 0.001 <8 years, p < 0.001 peripubertal, p < 0.05 post pubertal) but not between the young age groups. In those samples with biovular follicles the frequency was not significantly different between any of the groups. An antral follicle was observed in only one case <8 years of age (1.4 years) - 3.3%, in 2 in the peripubertal (8%), in 4 of the post pubertal (25%) and in 9 of the adult ovarian cases (10.6%). The higher observed frequency in the post pubertal ovary compared to the <8 year was significantly different (p < 0.05). Limitations, reasons for caution Only a small sample of the ovary cortex was examined for each individual, which may be more representative of the young ovary than the adult. It is not possible to predict potential function from this analysis. Wider implications of the findings Although follicle numbers in the paediatric ovary are high, follicles with structural anomalies such as biovular follicles are also more frequently observed in the paediatric ovary than in the adult. It appears that the changes at puberty may cause the loss of these abnormal follicles. Trial registration number Not Applicable

Analysis of serum reproductive hormones and ovarian genes in pubertal female goats

BackgroundAge at puberty is an important factor affecting goat fertility, with endocrine and genetic factors playing a crucial role in the onset of puberty. To better understand the relationship between endocrine and genetic factors and mechanisms underlying puberty onset in goats, reproductive hormone levels were analyzed by ELISA and ultraperformance liquid chromatography–multiple reaction monitoring–multistage/mass spectrometry and RNA sequencing was performed to analyze ovarian genes.ResultsSerum follicle stimulating hormone, luteinizing hormone, estradiol, 11-deoxycortisol, 11-deoxycorticosterone, corticosterone, cortisone, and cortisol levels were found to be higher but progesterone were lower in pubertal goats as compared to those in prepubertal goats (P < 0.05). A total of 18,139 genes were identified in cDNA libraries, and 75 differentially expressed genes (DEGs) were identified (|log2 fold change|≥ 1, P ≤ 0.05), of which 32 were significantly up- and 43 were down-regulated in pubertal goats. Gene ontology enrichment analyses indicated that DEGs were mainly involved in “metabolic process,” “signaling,” “reproduction,” and “growth.” Further, DEGs were significantly enriched in 91 Kyoto Encyclopedia of Genes and Genomes pathways, including estrogen signaling pathway, steroid hormone biosynthesis, and cAMP signaling pathway. Bioinformatics analysis showed that PRLR and THBS1 were highly expressed in pubertal ovaries, and ZP3, ZP4, and ASTL showed low expression, suggesting their involvement in follicular development and lutealization.ConclusionsTo summarize, serum hormone changes and ovarian DEGs expression were investigated in our study. Further studies are warranted to comprehensively explore the functions of DEGs in goat puberty.

Gestational Benzo[a]pyrene Exposure Destroys F1 Ovarian Germ Cells Through Mitochondrial Apoptosis Pathway and Diminishes Surviving Oocyte Quality.

Polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are products of incomplete combustion. In female mouse embryos primordial germ cells proliferate before and after arriving at the gonadal ridge around embryonic (E) 10 and begin entering meiosis at E13.5. Now oocytes, they arrest in the first meiotic prophase beginning at E17.5. We previously reported dose-dependent depletion of ovarian follicles in female mice exposed to 2 or 10 mg/kg-day BaP E6.5-15.5. We hypothesized that embryonic ovaries are more sensitive to gestational BaP exposure during the mitotic developmental window, and that this exposure results in persistent oxidative stress in ovaries and oocytes of exposed F1 female offspring. We orally dosed timed-pregnant female mice with 0 or 2 mg/kg-day BaP in oil from E6.5-11.5 (mitotic window) or E12.5-17.5 (meiotic window). Cultured E13.5 ovaries were utilized to investigate the mechanism of BaP-induced germ cell death. We observed statistically significant follicle depletion and increased ovarian lipid peroxidation in F1 pubertal ovaries following BaP exposure during either prenatal window. Culture of E13.5 ovaries with BaP induced germ cell DNA damage and release of cytochrome c from the mitochondria in oocytes, confirming that BaP exposure induced apoptosis via the mitochondrial pathway. Mitochondrial membrane potential, oocyte lipid droplet (LD) volume, and mitochondrial-LD colocalization were decreased and mitochondrial superoxide levels were increased in the MII oocytes of F1 females exposed gestationally to BaP. Results demonstrate similar sensitivity to germ cell depletion and persistent oxidative stress in F1 ovaries and oocytes following gestational BaP exposure during mitotic or meiotic windows.

Immunohistological expression of cytochrome P450 1A2 (CYP1A2) in the ovarian follicles of prepubertal and pubertal rat

Cytochrome P450 1A2 (CYP1A2) is a member of the cytochrome P450 superfamily enzymes in mammals and plays a major role in metabolizing endogenous hormones in the liver. In recent days, CYP1A2 expression has been found in not only the liver but also other tissues including the pancreas and lung. However, little information is available regarding the expression of CYP1A2 in the ovary, in spite of the facts that the ovarian follicle growth and atresia are tightly associated with controls of endocrine hormonal networks. Therefore, the expression of CYP1A2 in the ovaries of prepubertal and pubertal rats was investigated to assess its expression pattern and puberty-related alteration. It was demonstrated that the expression level of CYP1A2 was significantly (p < 0.01) higher in the pubertal ovaries than prepubertal counterparts. At the ovarian follicle level in both groups, whereas CYP1A2 expression was less detectable in the primordial, primary and secondary follicles, the strongly positive expression of CYP1A2 was localized in the granulosa cell layers in the antral and pre-ovulatory follicles. However, the ratio of CYP1A2-positive ovarian follicle was significantly (p < 0.01) higher in the ovary of pubertal group (73.1 ± 3.1%) than prepubertal one (41.0 ± 10.5%). During the Immunofluorescence, expression of CYP1A2 was mainly localized in Fas-positive follicles, indicating the atretic follicles. In conclusion, these results suggested that CYP1A2 expression was mainly localized at the atretic follicular cells and affected by the onset of puberty. Further study is still necessary but we hypothesize that CYP1A2 expresses in the atretic follicles to metabolize residue of the reproductive hormones. These findings may have important implications for the fields of reproductive biology of animals.

WHY THE RATE OF PCOS/PCOD INCREASE AT THE AGE OF MENARCHE

Polycystic ovary syndrome (PCOS) ratio increase at age of menarche worldwide.Objective: The objective is to determine the cause of PCOD/PCOS at the age of menarche.Methods: Age at menarche was compared with PCOS/PCOD, The data extracted by Medline, PubMed and Obsgyne online library that were queried for studies published between 1998 to 2021 by using specific MeSH terms. We reviewed 10 cross-sectional style analytical studies for the collection of data in this systematic article.Results: 10 studies conducted between the years 1998 to 2021 included in this systematic review. The age of menarche is between 10-18 years. Menarche is one of the major causes of PCOD/PCOS due to poor diagnosis of normal pubertal ovaries and polycystic ovaries. At the age of menarche the weight ratio is subjectively increased due to poor diet and then BMI increased. So PCOD/PCOS ratio increases day by day at the age of menarche.Conclusions: Age at menarche in women with PCOS is influenced by BMI and genetic Variants and poor diagnosis at ultrasound scan.

Gene Expression Profiling in Ovaries and Association Analyses Reveal HEP21 as a Candidate Gene for Sexual Maturity in Chickens.

Simple SummaryChicken meat and egg productions are essential for human beings. Sexual maturity is important for both egg production and meat flavor. It is necessary to elucidate the genetic mechanism of chicken sexual maturity. In current study, we used digital gene expression (DGE) RNA-sequencing analysis to investigate differential expression of genes in pre-pubertal and post-pubertal ovaries in two different sub-breeds of chicken with different onsets of sexual maturity. After the analysis of RNA-sequencing data, numerous differentially expressed genes were found in both comparisons (32 day old, early-sexual-maturity pre-laying hens (P-F-O1) vs. 103 day old early-sexual-maturity laying hens (P-F-O2), and 32 day old late-sexual-maturity pre-laying hens (L-F-O1) vs. 153 day old late-sexual-maturity pre-laying hens (L-F-O2)). With the bioinformatic analysis, hen egg protein 21 kDa (HEP21) was chosen as the candidate gene to conduct following experiment. The variations in HEP21 were screened and association analyses between rs315156783 and reproductive traits were investigated in fifth-generation Ningdu Yellow chickens from a closely bred population. These results demonstrated that HEP21 is a candidate gene for sexual maturity and ovary development in chickens. However, the underlying mechanism of how HEP21 regulates chicken sexual maturity needs further focused studies.The age of onset of sexual maturity is an important reproductive trait in chickens. In this study, we explored candidate genes associated with sexual maturity and ovary development in chickens. We performed DGE RNA-sequencing analyses of ovaries of pre-laying (P-F-O1, L-F-O1) and laying (P-F-O2, L-F-O2) hens of two sub-breeds of Ningdu Yellow chicken. A total of 3197 genes were identified in the two comparisons, and 966 and 1860 genes were detected exclusively in comparisons of P-F-O1 vs. P-F-O2 and L-F-O1 vs. L-F-O2, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that genes involved in transmembrane signaling receptor activity, cell adhesion, developmental processes, the neuroactive ligand–receptor interaction pathway, and the calcium signaling pathway were enriched in both comparisons. Genes on these pathways, including growth hormone (GH), integrin subunit beta 3 (ITGB3), thyroid stimulating hormone subunit beta (TSHB), prolactin (PRL), and transforming growth factor beta 3 (TGFB3), play indispensable roles in sexual maturity. As a gene unique to poultry, hen egg protein 21 kDa (HEP21) was chosen as the candidate gene. Differential expression and association analyses were performed. RNA-seq data and qPCR showed that HEP21 was significantly differentially expressed in pre-pubertal and pubertal ovaries. A total of 23 variations were detected in HEP21. Association analyses of single nucleotide polymorphisms (SNPs) in HEP21 and reproductive traits showed that rs315156783 was significantly related to comb height at 84 and 91 days. These results indicate that HEP21 is a candidate gene for sexual maturity in chickens. Our results contribute to a more comprehensive understanding of sexual maturity and reproduction in chickens.

The effect of GnRH antagonist cetrorelix on Wnt signaling members in pubertal and adult mouse ovaries.

Wide application of gonadotropin-releasing hormone (GnRH) agonists and antagonists for clinical purposes determines their effects on ovarian signaling pathways. Our study aimed to determine the localization, expression levels of Wnt signaling members in the pubertal and adult mouse ovary and the impact of GnRH antagonist cetrorelix on these signaling members. 0.5mg/kg of cetrorelix was injected to 3-and 6-week-old mice for 2 weeks. At the end of injection, ovaries from 5 (5Ce)- to 8-week (8Ce)-old mice were embedded in paraffin for immunohistochemistry and homogenized for western blot to compare with control (5C-8C) and sham groups (5S-8S). WNT2 and WNT4 showed higher expression in thecal and stromal cells in adult mouse ovaries and only WNT4 expression was affected by cetrorelix. FZD1 was localized mainly in oocytes of pubertal ovaries and granulosa cells and oocytes of adult ovaries. FZD1 was reduced by cetrorelix in pubertal ovaries. FZD4 was abundantly localized in thecal and stromal cells of all groups and protein level was not affected by cetrorelix. LRP-6 was expressed mainly in oocytes and stromal cells of pubertal, oocytes of adult ovaries and its expression was reduced by cetrorelix in adult ovaries. CTNNB1 intensity in granulosa cells was the lowest in pubertal and the highest in adult ovaries and its expression was decreased by cetrorelix in adult ovaries. Cetrorelix affected the expression of specific members of the Wnt signaling depending on the developmental stage of mice, pointing out its possible interaction with gonadotropins during pubertal and adult stages.

SAT-290 Expanded Phenotype Associated with RNF135 Intragenic Deletion in a Girl with Peripheral Precocious Puberty

Introduction: RNF135 gene is located within the 17q11.2 region and is often deleted in the NF1 contiguous microdeletion syndrome. Six individuals with suspected pathogenic mutations of RNF135 have been described with similar phenotypes including facial dysmorphism (downslanted palpebral fissures, elongated philtrum, broad nasal tip, thin upper lip, broad forehead), macrocephaly, tall stature, overgrowth, and learning disabilities. We present the first reported case of a patient with RNF135 intragenic deletion and precocious puberty. Clinical Case: Patient had ambiguous genitalia at birth, suspicious for CAH. Laboratory evaluation was normal. At age 2, she presented with breast and pubic hair development over the previous year. Leuprolide stimulation test was not consistent with central precocious puberty. Pelvic ultrasound revealed a paraovarian cyst that was drained at age 4 and pubertal development stabilized. At age 6, had progression of puberty with Tanner III breasts. MRI pelvis showed a large cyst of the left ovary and a post pubertal appearing uterus. Patient underwent left oophorectomy for numerous ovarian cysts and concern for progression of precocious puberty. Pathology showed a serous cystadenoma and pubertal ovary. At age 7 years 5 months, she had torsion of the right ovary and underwent laproscopic detorsion. Biopsy showed extensively hemorrhagic tissue with foci of ovarian stroma. Pubertal development was stable until age 7 years 10 months, when she presented with Tanner IV breasts and Tanner II pubic hair. MRI pelvis showed a multicystic right ovary with follicles. Since age 2, patient had tall stature, with height Z score ranging 3.45-4.47. She was diagnosed with focal epilepsy at age 3.5 years and had a PDA that was coiled at age 4. At age 7, chromosomal microarray showed an 88 base pair deletion at chromosome 17q11.2, including part of the RNF135 gene. Patient has dysmorphic facial features, including hypertelorism, upslanted palpebral fissures, bilateral epicanthal folds, flat nasal bridge, malar hypoplasia, high arched palate. Other dysmorphic body features include dolichocephaly and sandal gap between toes 1 and 2. Patient has fine motor, gross motor, and speech delays. Conclusion: Mutations in the RNF135 gene are rare, with phenotypes only described in 6 individuals to date. Our patient exhibits many phenotypic features seen in described patients with pathogenic variants in this gene, including dysmorphic facial features, developmental delay, and tall stature. This is the first reported case of a patient with RNF135 intragenic deletion presenting with peripheral precocious puberty, cystic ovaries, and focal epilepsy. This case describes several new clinical features that are thought to be associated with a rare RNF135-related overgrowth syndrome.

The ovarian germinal reserve and apoptosis-related proteins in the infant and adolescent human ovary

BackgroundNormal 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.ResultsOvarian 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.ConclusionsThese 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.

Regulation of neuroendocrine cells and neuron factors in the ovary by zinc oxide nanoparticles.

The pubertal period is an important window during the development of the female reproductive system. Development of the pubertal ovary, which supplies the oocytes intended for fertilization, requires growth factors, hormones, and neuronal factors. It has been reported that zinc oxide nanoparticles (ZnO NPs) cause cytotoxicity of neuron cells. However, there have been no reports of the effects of ZnO NPs on neuronal factors and neuroendocrine cells in the ovary (in vivo). For the first time, this in vivo study investigated the effects of ZnO NPs on gene and protein expression of neuronal factors and the population of neuroendocrine cells in ovaries. Intact NPs were detected in ovarian tissue and although ZnO NPs did not alter body weight, they reduced the ovary organ index. Compared to the control or ZnSO4 treatments, ZnO NPs treatments differentially regulated neuronal factor protein and gene expression, and the population of neuroendocrine cells. ZnO NPs changed the contents of essential elements in the ovary; however, they did not alter levels of the steroid hormones estrogen and progesterone. These data together suggest that intact ZnO NPs might pose a toxic effect on neuron development in the ovary and eventually negatively affect ovarian developmental at puberty.

The immature human ovary shows loss of abnormal follicles and increasing follicle developmental competence through childhood and adolescence

STUDY QUESTIONDo the ovarian follicles of children and adolescents differ in their morphology and in vitro growth potential from those of adults?SUMMARY ANSWERPre-pubertal ovaries contained a high proportion of morphologically abnormal non-growing follicles, and follicles showed reduced capacity for in vitro growth.WHAT IS KNOWN ALREADYThe pre-pubertal ovary is known to contain follicles at the early growing stages. How this changes over childhood and through puberty is unknown, and there are no previous data on the in vitro growth potential of follicles from pre-pubertal and pubertal girls.STUDY DESIGN, SIZE, DURATIONOvarian biopsies from five pre-pubertal and seven pubertal girls and 19 adult women were analysed histologically, cultured in vitro for 6 days, with growing follicles then isolated and cultured for a further 6 days.PARTICIPANTS/MATERIALS, SETTING, METHODSOvarian biopsies were obtained from girls undergoing ovarian tissue cryopreservation for fertility preservation, and compared with biopsies from adult women. Follicle stage and morphology were classified. After 6 days in culture, follicle growth initiation was assessed. The growth of isolated secondary follicles was assessed over a further 6 days, including analysis of oocyte growth.MAIN RESULTS AND THE ROLE OF CHANCEPre-pubertal ovaries contained a high proportion of abnormal non-growing follicles (19.4 versus 4.85% in pubertal ovaries; 4004 follicles analysed; P = 0.02) characterized by indistinct germinal vesicle membrane and absent nucleolus. Follicles with this abnormal morphology were not seen in the adult ovary. During 6 days culture, follicle growth initiation was observed at all ages; in pre-pubertal samples there was very little development to secondary stages, while pubertal samples showed similar growth activation to that seen in adult tissue (pubertal group: P = 0.02 versus pre-pubertal, ns versus adult). Isolated secondary follicles were cultured for a further 6 days. Those from pre-pubertal ovary showed limited growth (P < 0.05 versus both pubertal and adult follicles) and no change in oocyte diameter over that period. Follicles from pubertal ovaries showed increased growth; this was still reduced compared with follicles from adult women (P < 0.05) but oocyte growth was proportionate to follicle size.LIMITATIONS, REASONS FOR CAUTIONThese data derive from only a small number of ovarian biopsies, although large numbers of follicles were analysed. It is unclear whether the differences between groups are related to puberty, or just age.WIDER IMPLICATIONS OF THE FINDINGSThese findings show that follicles from girls of all ages can be induced to grow in vitro, which has important implications for some patients who are at high risk of malignant contamination of their ovarian tissue. The reduced growth of isolated follicles indicates that there are true intrafollicular differences in addition to potential differences in their local environment, and that there are maturational processes occurring in the ovary through childhood and adolescence, which involve the loss of abnormal follicles, and increasing follicle developmental competence.Study funding/competing interest(s)Funded by MRC grants G0901839 and G1100357. No competing interests.

The infant and pubertal human ovary: Balbiani's body-associated VASA expression, immunohistochemical detection of apoptosis-related BCL2 and BAX proteins, and DNA fragmentation

How do apoptosis-related BCL2 and BAX genes, known to regulate death or survival of germ cells in fetal and adult life, and germ-cell-specific VASA protein behave from birth to puberty in the human ovary? In resting primordial follicles in both infant and pubertal ovaries, BCL2 family members and germ-cell-specific VASA behave as in fetal life. After birth, once follicles leave the resting reserve to enter the growing follicular pool, detection of apoptosis-related genes moves from the germ cell to granulosa cells and VASA expression is lost. In the human ovary, around 85% of the 7 × 10⁶ potential oocytes produced at mid-gestation are lost before birth, an extra 10% before puberty, and loss continues throughout reproductive life until germinal exhaustion of the ovary. Oocyte loss is mainly driven through a balanced expression of BCL2 gene family members. Apoptosis-inducing BAX gene shows a sustained expression throughout fetal and adult life, whereas apoptosis-inhibiting BCL2 is detectable during the proliferative stage of primordial germ cells and oogonia in the fetal ovary and proliferation of granulosa cells in growing follicles in the adult ovary. The germ-cell marker VASA is detectable in the fetal ovary from early oogenesis and is conspicuously expressed in primordial follicles, where in late pregnancy it is associated with the Balbiani's vitelline space. VASA expression is not detectable in the adult ovary. This is a qualitative analysis involving infant/pubertal paraffin-embedded human ovaries screened for apoptosis-related proteins, DNA fragmentation and germ-cell identity. Ovaries from 13 patients ranging in age from 4 to 16 years, undergoing gynaecological surgical procedures due to benign pathology, were studied. Tissues were fixed in 10% formalin, paraffin-embedded and processed for immunohistochemistry to screen the temporal and cellular localization of germ-cell-specific VASA protein and BCL2 and BAX apoptosis-related proteins. In addition, a terminal deoxynucleotidyl transferase-mediated deoxiuridinetriphosphate nick-end labelling (TUNEL) assay was performed to detect DNA fragmentation. General histology and tissue integrity were assessed by haematoxylin-eosin staining. VASA showed a differential pattern of expression; in the resting primordial follicle reserve in infant and pubertal ovaries, it was associated with the Balbiani's body space in the germ cell. VASA remained detectable in primary follicles leaving the resting reserve, but once follicles entered the growing pool it became undetectable. This pattern of VASA expression is the same as in the fetal ovary. BAX was expressed both in the resting primordial reserve and in the pool of growing follicles, whereas BCL2 was detected only in granulosa cells in antral follicles in the growing pool. Apoptosis-related protein expression moved from the germ cell to the somatic stratum when primordial follicles left the resting reserve to enter the pool of growing follicles, irrespective of female age. Most TUNEL-positive cells were detected in the granulosa cells of antral follicles. No TUNEL-positive cells were found in resting primordial follicles. The study was limited by the qualitative nature of the immuno-histochemical analysis and the TUNEL assay. The results neither quantify the levels of germ-cell death nor exclude other concurrent cell death mechanisms that could act in the regulation of female germ-cell number. This study provides missing knowledge about apoptosis and germ-cell-specific VASA expression in the human ovary between birth and puberty and the participation of BCL2 and BAX genes in the balance between death and survival throughout female germ-line development. Intracellular localization of VASA in resting follicles emerges as a possible marker with prognostic value that needs further investigation, especially in infant patients entering ovarian cryo-preservation programmes. This knowledge will be valuable in optimizing the rescue and clinical use of germ cells to restore fertility in women. No external funding was obtained for this study. The authors have no conflicts of interest to declare.

Study of Neonatal Exposure to Estrogenic and Androgenic Endocrine Disruptors by Normal-Phase HPLC

Neonatal exposure to estrogenic and androgenic endocrine disruptors induces developmental abnormalities in the female reproductive system. To investigate whether neonatal exposure affects oogenesis in juvenile and pubertal ovary, Sprague-Dawley rat pups were given estrogen or various endocrine disruptors by a single injection on the day of birth at concentrations ranging between 2 mM to 40 mM, and sacrificed on day 21 (juvenile) or 50 (puberty). The ovaries were weighed and examined histologically at each stage. Further, 17 β-estradiol synthesis of oogenesis was analyzed using normal-phase high-performance liquid chromatography. Neonatal exposure to 17 β-estradiol, diethylstilbestrol and estrogen receptor inhibitors significantly reduced ovary weights, although estrogen receptor inhibitors were completely restored by 17 β-estradiol synthesis during puberty. Keywords: Endocrine disruptor, neonatal exposure, ovary, oogenesis, 17 β-estradiol, estrogen receptor.

Gonadotropic regulation of circadian clockwork in rat granulosa cells

The circadian clock is responsible for the generation of circadian rhythms in hormonal secretion and metabolism. These peripheral clocks could be reset by various cues in order to adapt to environmental variations. The ovary can be characterized as having highly dynamic physiology regulated by gonadotropins. Here, we aimed to address the status of circadian clock in the ovary, and to explore how gonadotropins could regulate clockwork in granulosa cells (GCs). To this end, we mainly utilized the immunohistochemistry, RT-PCR, and real-time monitoring of gene expression methods. PER1 protein was constantly abundant across the daily cycle in the GCs of immature ovaries. In contrast, PER1 protein level was obviously cyclic through the circadian cycle in the luteal cells of pubertal ovaries. In addition, both FSH and LH induced Per1 expression in cultured immature and mature GCs, respectively. The promoter analysis revealed that the Per1 expression was mediated by the cAMP response element binding protein. In cultured transgenic GCs, both FSH and LH also induced the circadian oscillation of Per2. However, the Per2 oscillation promoted by FSH quickly dampened within only one cycle, whereas the Per2 oscillation promoted by LH was persistently maintained. Collectively, these findings strongly suggest that both FSH and LH play an important role in regulating circadian clock in the ovary; however, they might exert differential actions on the clockwork in vivo due to each specific role within ovarian physiology.

The effects of perinatal/juvenile methoxychlor exposure on adult rat nervous, immune, and reproductive system function.

In order to address data gaps identified by the NAS report Pesticides in the Diets of Infants and Children, a study was performed using methoxychlor (MXC). Female rats were gavaged with MXC at 0, 5, 50, or 150 mg/kg/day for the week before and the week after birth, whereupon the pups were directly dosed with MXC from postnatal day (pnd) 7. Some dams were killed pnd7 and milk and plasma were assayed for MXC and metabolites. For one cohort of juveniles, treatment stopped at pnd21; a modified functional observational battery was used to assess neurobehavioral changes. Other cohorts of juveniles were dosed until pnd42 and evaluated for changes to the immune system and for reproductive toxicity. Dose-dependent amounts of MXC and metabolites were present in milk and plasma of dams and pups. The high dose of MXC reduced litter size by approximately 17%. Ano-genital distance was unchanged, although vaginal opening was accelerated in all treated groups, and male prepuce separation was delayed at the middle and high doses by 8 and 34 days, respectively. In the neurobehavioral evaluation, high-dose males were more excitable, but other changes were inconsistent and insubstantial. A decrease in the antibody plaque-forming cell response was seen in males only. Adult estrous cyclicity was disrupted at 50 and 150 MXC, doses which also showed reduced rates of pregnancy and delivery. Uterine weights (corrected for pregnancy) were reduced in all treated pregnant females. High-dose males impregnated fewer untreated females; epididymal sperm count and testis weight were reduced at the high, or top two, doses, respectively. All groups of treated females showed uterine dysplasias and less mammary alveolar development; estrous levels of follicle stimulating hormone were lower in all treated groups, and estrus progesterone levels were lower at 50 and 150 MXC, attributed to fewer corpora lutea secondary to ovulation defects. These data collectively show that the primary adult effects of early exposure to MXC are reproductive, show that 5 mg/kg/day is not a NO(A)EL in rats with this exposure paradigm (based on changes in day of vaginal opening, pubertal ovary weights, adult uterine and seminal vesicle weights, and female hormone data) and imply that the sites of action are both central and peripheral.

Developmental expression of heat shock protein 60 (HSP60) in the rat testis and ovary

Heat shock protein 60 (HSP60), a member of the chaperonin family, has an essential role in mediating correct folding of nuclear encoded proteins imported to mitochondria. We have investigated immunocytochemical expression of HSP60 in developing fetal, newborn, postnatal, and pubertal testis and ovary, and in the adult ovary of the rat. In the fetal gonads, HSP60 was expressed in the germ cells organized into sex cords and in the developing Leydig cells of the testis. In the pubertal testis, Leydig cells were strongly, spermatogonia and premeiotic spermatocytes moderately labeled, spermatids unlabeled. In the postnatal ovary, oocytes at all stages of folliculogenesis were positive for HSP60. In the pubertal ovary, glandular theca cells, and in the mature ovary, also the cells of the corpora lutea exhibited intense cytoplasmic labeling. At the electron microscopic level, immunogold particles were localized in the mitochondrial matrix, and in the Western blot analysis the antibody detected one single band of 60 kDa. Anti-HSP60 labeling in male and female sex steroid producing cells and their progenitors seems to be coordinated with the functional differentiation of these endocrine cells of the gonad. In the oocytes, a key element required for proper folding of imported mitochondrial proteins seems to be constitutively expressed throughout folliculogenesis. However, the data suggest that in the male germ cells mitochondrial chaperonin HSP60 is either not needed during the haploid phase of spermatogenesis or its level becomes extensively reduced and therefore undetectable by the methods used in the study.

Developmental expression of heat shock protein 60 (HSP60) in the rat testis and ovary

Heat shock protein 60 (HSP60), a member of the chaperonin family, has an essential role in mediating correct folding of nuclear encoded proteins imported to mitochondria. We have investigated immunocytochemical expression of HSP60 in developing fetal, newborn, postnatal, and pubertal testis and ovary, and in the adult ovary of the rat. In the fetal gonads, HSP60 was expressed in the germ cells organized into sex cords and in the developing Leydig cells of the testis. In the pubertal testis, Leydig cells were strongly, spermatogonia and premeiotic spermatocytes moderately labeled, spermatids unlabeled. In the postnatal ovary, oocytes at all stages of folliculogenesis were positive for HSP60. In the pubertal ovary, glandular theca cells, and in the mature ovary, also the cells of the corpora lutea exhibited intense cytoplasmic labeling. At the electron microscopic level, immunogold particles were localized in the mitochondrial matrix, and in the Western blot analysis the antibody detected one single band of 60 kDa. Anti-HSP60 labeling in male and female sex steroid producing cells and their progenitors seems to be coordinated with the functional differentiation of these endocrine cells of the gonad. In the oocytes, a key element required for proper folding of imported mitochondrial proteins seems to be constitutively expressed throughout folliculogenesis. However, the data suggest that in the male germ cells mitochondrial chaperonin HSP60 is either not needed during the haploid phase of spermatogenesis or its level becomes extensively reduced and therefore undetectable by the methods used in the study.

Developmental changes in mullerian inhibiting substance in the cynomolgus monkey, Macaca fascicularis.

Mullerian inhibiting substance (MIS) is a glycoprotein hormone produced in Sertoli cells of the fetal and postnatal testis, and granulosa cells of the pubertal ovary. We examined MIS expression in a nonhuman primate, the cynomolgus macaque monkey (Macaca fascicularis), to define an animal model for studying MIS gene regulation. Changes in testicular MIS mRNA with age were assessed by in situ hybridization of prepubertal to adult testes, Northern analysis of pubertal and adult specimens, and determination of serum MIS concentrations from infancy to adulthood. We found that MIS expression was highest in the youngest animals and decreased progressively with increasing age. Serum MIS concentrations correlated inversely with increasing age (r = -0.74), body weight (r = -0.79), and testicular volume (r = -0.73), but not with testosterone levels (r = -0.35). The mean MIS concentrations +/- SEM for the four developmental age groups were 270.6 +/- 23.8 (infants), 195.5 +/- 18.5 (juveniles), 102.7 +/- 28.4 (peripubertals), and 51.6 +/- 7.1 (adults). This study confirms that nonhuman primate and human MIS are highly homologous and have similar developmental patterns. The normative data for serum MIS concentrations in cynomolgus monkeys at different ages and developmental stages will be invaluable for further work examining MIS regulation.

Menstrual irregularities in adolescents: hormonal pattern and ovarian morphology.

The endocrine pattern and ovarian characteristics of 110 healthy adolescents with menstrual irregularities were investigated during the early follicular and premenstrual phases and were compared to those of 14 adolescents with regular menstrual cycles and 20 adults. Over a period of six gynecological years a low ovulation rate (49%) was found in the group of subjects with irregular cycles and regular ovulation was noted in only a few subjects. Slight differences in endocrine pattern and ovarian morphology were observed between the group of adolescents with regular cycles and the group of adults. In contrast, adolescents with irregular menses had higher mean values of luteinizing hormone (LH), testosterone (T), and androstenedione (A) in comparison with the other two groups both in follicular and premenstrual phases. Nearly 35% of the subjects with irregular cycles had levels of T, A and LH which were higher than the upper limit of the adult normal range. Lower progesterone (P), 17P and oestradiol values were observed in the premenstrual phase. Within the group of subjects with irregular menses, LH levels were higher in anovulatory than in ovulatory cycles, in both phases of the cycle, while T and A levels were higher and prolactin levels were lower in the premenstrual phase of anovulatory cycles. Unlike irregular anovulatory cycles, irregular ovulatory cycles showed a hormonal pattern similar to that found in the adult group. By ultrasound evaluation, a high percentage of subjects with irregular menses had multicystic ovaries (57.9%) and the mean (+/- SEM) ovarian volume was higher (10.6 +/- 0.5 cm3) than that found in adolescents with regular menses (6.7 +/- 0.8 cm3) and in the adult group (7.7 +/- 0.3 cm3). With the increase in frequency and continuity of ovulation an improvement in the direction of adult volume and ovarian structure was observed. Besides the endocrine similarity the data emphasize the striking similarity, already documented by histological studies, between pubertal ovaries and those seen in micropolycystic ovary syndrome. These endocrine and ovarian characteristics are typical of a large number of adolescents with irregular menstrual cycles: these features may be representative of a developmental step toward adult normality, although the possibility of a pathological evolution for some subjects cannot be excluded.