Neonatal Rat Ovaries Research Articles

The effect of bisphenol A on the Notch (Notch2 and Jagged2) signaling pathway in the follicular development of the neonatal rat ovary

ABSTRACT The formation of primordial follicles determines the pool size of follicles in the ovary, and is crucial for female reproductivity. Oocyte nest breakdown, and the formation of primordial follicles, largely depend upon the communication between oocytes and the surrounding pregranulosa cells. The neurogenic locus notch homolog protein (Notch) signaling pathway is the key player for this cell-to-cell communication, and is responsible for primordial folliculogenesis. However, different endocrine disruptors, including bisphenol A (BPA; a plasticizer and a constituent of reusable plastic containers) may affect the Notch signaling pathway, and might induce ovary dysfunction via Notch signaling. Consequently, we investigated the possible influence of BPA treatment on the proportional distribution of the follicular stages, follicle numbers, levels of apoptosis, and on Notch2 and Jagged2 expressions in the ovary. BPA was administered at doses of either 50 µg/kg/day or 50 mg/kg/day, at different time intervals, during neonatal and fetal periods in vivo. After collecting the ovaries from the various experimental groups, follicles were counted, and frequency of apoptosis was determined by TUNEL assay. In addition, Notch2 and Jagged2 expressions were assessed by immunohistochemical staining and qPCR. In summary, BPA treatment affected the follicle numbers and apoptosis level, and Notch2 and Jagged2 expressions varied with follicular stage. It was also observed that these parameters were dose and time dependent with respect to BPA exposure.

Expression patterns of p38αMAPK during follicular development in the ovaries of neonatal rats

The p38αMAPK signaling pathway plays a critical role in female reproduction, but an understanding of its expression in rats remains elusive. This study was carried out to investigate the temporal and spatial expression of p38αMAPK and p-p38αMAPK. Ovarian tissue samples were collected from 2-, 4-, 8-, 12-, 16-, 20- and 30-day-old female rats. Western blotting was used to examine the relative expression of p38αMAPK and p-p38αMAPK in ovarian tissue, and subcellular localization was examined using immunohistochemistry of the rat ovaries at different ages of postpartum. The immunohistochemical results showed that p38αMAPK and p-p38αMAPK were widely expressed in the rat ovaries, mainly localized in the follicle cells and granulosa cells. The expression of p38αMAPK was relatively stable for the different stages of oocytes, whereas the expression of p-p38αMAPK gradually increased. At different stages of granulosa cells, the expression of p38αMAPK was also relatively stable, and the p-p38αMAPK expression showed an upward trend during follicular development. Western blotting revealed that the expression of p38αMAPK in the ovaries was relatively stable, where as p-p38αMAPK expression initially exhibited an increasing trend and subsequently decreased, with a maximum at day 20. The expression patterns of p38αMAPK and p-p38αMAPK in the rat ovaries indicate their possible involvement in folliculogenesis. Taken together, the stage- and cell-specific expression of p-p38αMAPK in rat ovaries indicated that p-p38αMAPK might play a vital role during rat follicular development.

Estrogenic Compounds Impair Primordial Follicle Formation by Inhibiting the Expression of Proapoptotic Hrk in Neonatal Rat Ovary.

Exposure to endocrine-disrupting chemicals (EDCs) during fetal and neonatal periods can have toxic effects that are irreversible and last a lifetime. However, the mechanism underlying this phenomenon is still unknown. Here, we show the effect of 17alpha-ethynyl estradiol (EE) on the development of the primordial follicle during early ovarian development in female rats. Microarray analysis revealed the down-regulation of Hrk, an activator of apoptosis, in neonatal ovaries exposed to EE. Real-time PCR analysis also showed a decrease of Hrk mRNA expression in ovaries treated with EE both in vitro and in neonatal rats. An immunostaining assay showed that HRK protein and cleaved caspase 3 colocalize in the oocytes at Postnatal Day 1 (PND1). The EE-exposed ovaries had a reduced number of oocytes positive for TUNEL staining compared to control ovaries at PND1. Abnormal follicle formation of EE-exposed ovaries was observed at PND7 and PND21. A TUNEL staining assay revealed that Hrk depletion reduced the number of apoptotic oocytes. In addition, down-regulation of Hrk mRNA expression was observed in ovaries treated with other estrogenic chemicals. We propose a model in which EE inhibits oocyte apoptosis in the neonatal ovary by suppressing the expression of Hrk, thereby disrupting follicle formation and ovary function.

Phosphodiesterases in the rat ovary: effect of cAMP in primordial follicles.

Phosphodiesterases (PDEs) are important regulators of the intracellular cAMP concentration, which is a central second messenger that affects a multitude of intracellular functions. In the ovaries, cAMP exerts diverse functions, including regulation of ovulation and it has been suggested that augmented cAMP levels stimulate primordial follicle growth. The present study examined the gene expression, enzyme activity and immunolocalization of the different cAMP hydrolysing PDEs families in the rat ovary. Further, the effect of PDE4 inhibition on primordial follicle activation in cultured neonatal rat ovaries was also evaluated. We found varied expression of all eight families in the ovary with Pde7b and Pde8a having the highest expression each accounting for more than 20% of the total PDE mRNA. PDE4 accounted for 15-26% of the total PDE activity. Immunoreactive PDE11A was found in the oocytes and PDE2A in the corpora lutea. Incubating neonatal rat ovaries with PDE4 inhibitors did not increase primordial follicle activation or change the expression of the developing follicle markers Gdf9, Amh, Inha, the proliferation marker Mki67 or the primordial follicle marker Tmeff2. In addition, the cAMP analogue 8-bromo-cAMP did not increase AKT1 or FOXO3A phosphorylation associated with follicle activation or increase the expression of Kitlg known to be associated with follicle differentiation but did increase the Tmeff2, Mki67 and Inha expression in a dose-dependent manner. In conclusion, this study shows that both Pde7b and Pde8a are highly expressed in the rodent ovary and that PDE4 inhibition does not cause an increase in primordial follicle activation.

Tamoxifen prevents apoptosis and follicle loss from cyclophosphamide in cultured rat ovaries.

Recent studies documented that the selective estrogen receptor modulator tamoxifen prevents follicle loss and promotes fertility following in vivo exposure of rodents to irradiation or ovotoxic cancer drugs, cyclophosphamide and doxorubicin. In an effort to characterize the ovarian-sparing mechanisms of tamoxifen in preantral follicle classes, cultured neonatal rat ovaries (Day 4, Sprague Dawley) were treated for 1-7 days with active metabolites of cyclophosphamide (i.e., 4-hydroxycyclophosphamide; CTX) (0, 1, and 10 μM) and tamoxifen (i.e., 4-hydroxytamoxifen; TAM) (0 and 10 μM) in vitro, and both apoptosis and follicle numbers were measured. CTX caused marked follicular apoptosis and follicular loss. TAM treatment decreased follicular loss and apoptosis from CTX in vitro. TAM alone had no effect on these parameters. IGF-1 and IGF-1 receptor were assessed in ovarian tissue showing no impact of TAM or CTX on these endpoints. Targeted mRNA analysis during follicular rescue by TAM revealed decreased expression of multiple genes related to inflammation, including mediators of lipoxygenase and prostaglandin production and signaling (Alox5, Pla2g1b, Ptgfr), cytokine binding (Il1r1, Il2rg ), apoptosis (Tnfrsf1a), second messenger signaling (Mapk1, Mapk14, Plcg1), as well as tissue remodeling and vasodilation (Bdkrb2, Klk15). The results suggest that TAM protects the ovary from CTX-mediated toxicity through direct ovarian actions that oppose follicular loss.

Impact of obesity on 7,12-dimethylbenz[a]anthracene-induced altered ovarian connexin gap junction proteins in female mice

The ovarian gap junction proteins alpha 4 (GJA4 or connexin 37; CX37), alpha 1 (GJA1 or connexin 43; CX43) and gamma 1 (GJC1 or connexin 45; CX45) are involved in cell communication and folliculogenesis. 7,12-dimethylbenz[a]anthracene (DMBA) alters Cx37 and Cx43 expression in cultured neonatal rat ovaries. Additionally, obesity has an additive effect on DMBA-induced ovarian cell death and follicle depletion, thus, we investigated in vivo impacts of obesity and DMBA on CX protein levels. Ovaries were collected from lean and obese mice aged 6, 12, 18, or 24 wks. A subset of 18 wk old mice (lean and obese) were dosed with sesame oil or DMBA (1mg/kg; ip) for 14days and ovaries collected 3days thereafter. Cx43 and Cx45 mRNA and protein levels decreased (P<0.05) after 18 wks while Cx37 mRNA and protein levels decreased (P<0.05) after 24 wks in obese ovaries. Cx37 mRNA and antral follicle protein staining intensity were reduced (P<0.05) by obesity while total CX37 protein was reduced (P<0.05) in DMBA exposed obese ovaries. Cx43 mRNA and total protein levels were decreased (P<0.05) by DMBA in both lean and obese ovaries while basal protein staining intensity was reduced (P<0.05) in obese controls. Cx45 mRNA, total protein and protein staining intensity level were decreased (P<0.05) by obesity. These data support that obesity temporally alters gap junction protein expression and that DMBA-induced ovotoxicity may involve reduced gap junction protein function.

The effects of bisphenol A in in vitro neonatal rat ovary

The effects of bisphenol A in in vitro neonatal rat ovary

The effect of bisphenol A on Notch signaling pathway in the follicular development of neonatal rat ovary

The effect of bisphenol A on Notch signaling pathway in the follicular development of neonatal rat ovary

Simultaneous isolation of mRNA and native protein from minute samples of cells.

Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization using different protein assays.

Acute 7,12-dimethylbenz[a]anthracene exposure causes differential concentration-dependent follicle depletion and gene expression in neonatal rat ovaries

Chronic exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA), generated during combustion of organic matter including cigarette smoke, depletes all ovarian follicle types in the mouse and rat, and in vitro models mimic this effect. To investigate the mechanisms involved in follicular depletion during acute DMBA exposure, two concentrations of DMBA at which follicle depletion has (75nM) and has not (12.5nM) been observed were investigated. Postnatal day four F344 rat ovaries were maintained in culture for four days before a single exposure to vehicle control (1% DMSO; CT) or DMBA (12nM; low-concentration or 75nM; high-concentration). After four or eight additional days of culture, DMBA-induced follicle depletion was evaluated via follicle enumeration. Relative to control, DMBA did not affect follicle numbers after 4days of exposure, but induced large primary follicle loss at both concentrations after 8days; while, the low-concentration DMBA also caused secondary follicle depletion. Neither concentration affected primordial or small primary follicle number. RNA was isolated and quantitative RT-PCR performed prior to follicle loss to measure mRNA levels of genes involved in xenobiotic metabolism (Cyp2e1, Gstmu, Gstpi, Ephx1), autophagy (Atg7, Becn1), oxidative stress response (Sod1, Sod2) and the phosphatidylinositol 3-kinase (PI3K) pathway (Kitlg, cKit, Akt1) 1, 2 and 4days after exposure. With the exception of Atg7 and cKit, DMBA increased (P<0.05) expression of all genes investigated. Also, BECN1 and pAKTThr308 protein levels were increased while cKIT was decreased by DMBA exposure. Taken together, these results suggest an increase in DMBA bioactivation, add to the mechanistic understanding of DMBA-induced ovotoxicity and raise concern regarding female low concentration DMBA exposures.

SAS1B protein [ovastacin] shows temporal and spatial restriction to oocytes in several eutherian orders and initiates translation at the primary to secondary follicle transition

Sperm Acrosomal SLLP1 Binding (SAS1B) protein (ovastacin) is an oolemmal binding partner for the intra-acrosomal sperm protein SLLP1. Immunohistochemical localization revealed that SAS1B translation is restricted among adult tissues to the ovary and oocytes, SAS1B appearing first in follicles at the primary-secondary transition. Quiescent oocytes within primordial follicles and primary follicles did not stain for SAS1B. Examination of neonatal rat ovaries revealed SAS1B expression first as faint signals in postnatal day 3 oocytes, with SAS1B protein staining intensifying with oocyte growth. Irrespective of animal age or estrus stage, SAS1B was seen only in oocytes of follicles that initiated a second granulosa cell layer. The precise temporal and spatial onset of SAS1B expression was conserved in adult ovaries in seven eutherian species, including nonhuman primates. Immunoelectron micrographs localized SAS1B within cortical granules in MII oocytes. A population of SAS1B localized on the oolemma predominantly in the microvillar region anti-podal to the nucleus in ovulated MII rat oocytes and on the oolemma in macaque GV oocytes. The restricted expression of SAS1B protein in growing oocytes, absence in the ovarian reserve, and localization on the oolemma suggest this zinc metalloprotease deserves consideration as a candidate target for reversible female contraceptive strategies.

7,12-Dimethylbenz[a]anthracene exposure induces the DNA repair response in neonatal rat ovaries

7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all stages of development. This study investigated DMBA-induced DNA double strand break (DSB) formation with subsequent activation of the ovarian DNA repair response in models of pre-antral or pre-ovulatory follicle loss. Postnatal day (PND) 4 Fisher 344 (F344) rat ovaries were cultured for 4days followed by single exposures of vehicle control (1% DMSO) or DMBA (12.5nM or 75nM) and maintained in culture for 4 or 8days. Alternately, PND4 F344 rat ovaries were exposed to 1μM DMBA at the start of culture for 2days. Total RNA or protein was isolated, followed by qPCR or Western blotting to quantify mRNA or protein level, respectively. γH2AX and phosphorylated ATM were localized and quantified using immunofluorescence staining. DMBA exposure increased caspase 3 and γH2AX protein. Additionally, DMBA (12.5nM and 1μM) increased levels of mRNA encoding Atm, Xrcc6, Brca1 and Rad51. In contrast, Parp1 mRNA was decreased on d4 and increased on d8 of DMBA exposure, while PARP1 protein increased after 8days of DMBA exposure. Total ATM increased in a concentration-dependent temporal pattern (75nM d4; 12.5nM d8), while pATM was localized in large primary and secondary follicles and increased after 8days of 75nM DMBA exposure compared to both control and 12.5nM DMBA. These findings support that, despite some concentration effects, DMBA induces ovarian DNA damage and that DNA repair mechanisms are induced as a potential mechanism to prevent follicle loss.

Gene bionetworks that regulate ovarian primordial follicle assembly

BackgroundPrimordial follicle assembly is the process by which ovarian primordial follicles are formed. During follicle assembly oocyte nests break down and a layer of pre-granulosa cells surrounds individual oocytes to form primordial follicles. The pool of primordial follicles formed is the source of oocytes for ovulation during a female’s reproductive life.ResultsThe current study utilized a systems approach to detect all genes that are differentially expressed in response to seven different growth factor and hormone treatments known to influence (increase or decrease) primordial follicle assembly in a neonatal rat ovary culture system. One novel factor, basic fibroblast growth factor (FGF2), was experimentally determined to inhibit follicle assembly. The different growth factor and hormone treatments were all found to affect similar physiological pathways, but each treatment affected a unique set of differentially expressed genes (signature gene set). A gene bionetwork analysis identified gene modules of coordinately expressed interconnected genes and it was found that different gene modules appear to accomplish distinct tasks during primordial follicle assembly. Predictions of physiological pathways important to follicle assembly were validated using ovary culture experiments in which ERK1/2 (MAPK1) activity was increased.ConclusionsA number of the highly interconnected genes in these gene networks have previously been linked to primary ovarian insufficiency (POI) and polycystic ovarian disease syndrome (PCOS). Observations have identified novel factors and gene networks that regulate primordial follicle assembly. This systems biology approach has helped elucidate the molecular control of primordial follicle assembly and provided potential therapeutic targets for the treatment of ovarian disease.

Glutathione S-transferase class mu regulation of apoptosis signal-regulating kinase 1 protein during VCD-induced ovotoxicity in neonatal rat ovaries

4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied. Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30μM) for 2–8days; 2) VCD (30μM) for 2days, followed by incubation in control media for 4days (acute VCD exposure); or 3) LY294002 (20μM) for 6days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P<0.05) after 6days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P<0.05) relative to control after 6days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P<0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P<0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1.

Role of Glutathione S-Transferase Class Mu During VCD-Induced Ovotoxicity in Neonatal Rat Ovaries.

4-vinylcyclohexene diepoxide (VCD) is an occupational chemical that selectively destroys ovarian small pre-antral follicles in both rats and mice via apoptosis. Glutathione S-transferase (GST) enzymes catalyze glutathione (GSH) conjugation to xenobiotics. In cultured neonatal mouse ovaries exposed to VCD, mRNA encoding the GST isoform mu (GSTm) increased (d4) prior to the onset of follicle loss (d6). GST protein can also regulate cell signaling, and in extra-ovarian tissues GSTM prevents the action of apoptosis signal-regulating kinase 1 (ASK1) through formation of a protein complex. Upon xenobiotic exposure, this protein complex dissociates resulting in activation of pro-apoptotic signaling by ASK1. This study was designed to investigate an involvement of GSTm in VCD-induced ovotoxicity in rats. Post-natal day 4 (PND4) rat ovaries were incubated in control medium ± VCD (30 μM) for 2, 4, 6 or 8 d. Following culture, total RNA or protein were isolated (n=3; 10-20 ovaries per pool). GSTm mRNA and protein levels were determined using quantitative RT-PCR and Western blotting, respectively. To assess the presence of an GSTM:ASK1 protein complex, immunoprecipitation using an anti-ASK1 primary antibody was followed by Western blotting to measure GSTM protein level. There was no effect of VCD exposure on Gstm mRNA expression at any time point. Despite lack of a transcriptional effect GSTM protein was increased (P < 0.05) by VCD exposure after 6 and 8 d, relative to control. A GSTM:ASK1 protein complex was detected in the rat ovary. Relative to control, there was no effect of VCD on the GSTM:ASK1 protein complex after 4 d of exposure. GSTM-bound to ASK1 increased (P < 0.05) after 6 d of VCD exposure, when follicle loss is underway. Because there was no increase in the amount of GSTM protein unbound to ASK1, this indicates that the increase in total GSTM reflected an increase in the ASK1-bound form. These findings support that GSTM protein is involved in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1. (Supported by ES016818 to AFK, ES09246 to PBH).

Parabens inhibit the early phase of folliculogenesis and steroidogenesis in the ovaries of neonatal rats

Parabens are widely used as anti-microbial agents in the cosmetic and pharmaceutical industries. Recently, parabens have been shown to act as xenoestrogens, a class of endocrine disruptors. In the present study, 55 female pups were given daily subcutaneous injections of methyl-, propyl-, and butyl-paraben or 17beta-estradiol (E2) during neonatal Day 1-7. The ovaries were excised on postnatal Day 8, then fixed and stained with hematoxylin and eosin for histological analysis. The follicles were counted and classified as being in the primordial, early primary, or primary stages. The number of primordial follicles increased while early primary follicles decreased at the high doses of propyl- and butyl-paraben. The levels of anti-Mullerian hormone (AMH) and Foxl2 mRNA increased by propyl- and butyl-parabens whereas kit ligand/stem cell factor (KITL) expression was up regulated only by butyl-paraben. The mRNA levels of StAR and Cyp11a1 were significantly decreased after treatment with methyl-, propyl-, and butyl-parabens. Consistent with its use as a positive control, E2 regulated the expression of KITL, StAR, and Cyp11a1 genes, but surprisingly did not affect AMH and Foxl2 levels. Thus, E2 and parabens had different effects on the regulation of folliculogenic and steroidogenic genes, demonstrating the estrogenic and nonestrogenic properties of parabens in the ovary. Taken together, our data show that parabens stimulated AMH mRNA expression and consequently inhibited the early phase of folliculogenesis in the ovaries of neonatal female rat. The levels of steroidogenic enzymes, indicators of follicle differentiation, appeared to be regulated by parabens through inhibition of their transcriptional repressor, Foxl2.

Parabens disrupt folliculogenesis by induction of anti-Mullerian hormone gene expression and regulate steroidogenesis in the ovaries of neonatal rats

Parabens disrupt folliculogenesis by induction of anti-Mullerian hormone gene expression and regulate steroidogenesis in the ovaries of neonatal rats

4-vinylcyclohexene diepoxide: a model chemical for ovotoxicity

The occupational chemical 4-vinylcyclohexene diepoxide (VCD) has been shown to cause selective destruction of ovarian small pre-antral (primordial and primary) follicles in rats and mice by accelerating the natural, apoptotic process of atresia. Chemicals that destroy primordial follicles are of concern to women because exposure can result in premature ovarian failure (early menopause). In`itial studies using in vivo exposure of rats determined that VCD specifically targets primordial and primary (small pre-antral) follicles and that repeated dosing is required. Through a method of isolation of ovarian small follicles, biochemical and molecular studies determined that intracellular pro-apoptotic pathways are activated following VCD dosing in rats. Subsequently an in vitro system using cultured whole neonatal rat ovaries was developed to provide more mechanistic information. That approach was used to demonstrate that the cell survival c-kit//kit ligand signaling pathway is the direct target for VCD-induced ovotoxicity. Specifically, VCD directly interacts with the oocyte-associated c-kit receptor to inhibit its autophosphorylation, and thereby impair oocyte viability. The cellular and molecular approach developed to determine these findings is described in this article.

Cell-specific expression and immunolocalization of nitric oxide synthase isoforms and the related nitric oxide/cyclic GMP signaling pathway in the ovaries of neonatal and immature rats

The present study is designed to investigate the cellular expressions and immunolocalizations of three different nitric oxide synthase (NOS) isoforms and the related nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway in the ovaries of neonatal and immature rats. The ovaries were obtained from ICR (Institute for Cancer Research) female Sprague-Dawley rats at postnatal days 1, 5, 7, 10, and 19. Then we carried out the histologic examination, immunohistochemistry, measurement of NOS activity, and modifications within the NO/cGMP pathway. During postnatal days 1, 5, 7, 10, and 19, all three isoforms of NOS were mainly localized to the oocytes and expressed as a gradual increase in granulosa cells and theca cells within the growing follicle. The ovarian total NOS activities and NO levels were increased at postnatal days 7 and 10 compared with other days. Our findings suggest that the locally produced NO and the NO/NOS signaling systems are involved in the follicular development to puberty.

Distribution and responsiveness of rat anti-Müllerian hormone during ovarian development and VCD-induced ovotoxicity

Anti-Müllerian hormone (AMH) is produced by granulosa cells in primary to small antral follicles of the adult ovary and helps maintain primordial follicles in a dormant state. The industrial chemical, 4-vinylcyclohexene diepoxide (VCD) causes specific ovotoxicity in primordial and small primary follicles of mice and rats. Previous studies suggest that this ovotoxicity involves acceleration of primordial to primary follicle recruitment via interactions with the Kit/Kit ligand signaling pathway. Because of its accepted role in inhibiting primordial follicle recruitment, the present study was designed to investigate a possible interaction between AMH and VCD-induced ovotoxicity. Protein distribution of AMH was compared in neonatal and adult F344 rat ovaries. AMH protein was visualized by immunofluorescence microscopy in large primary and secondary follicles of the adult ovary, but in small primary follicles in neonatal rat ovaries. In cultured postnatal day (PND) 4 F344 rat ovaries, VCD exposure (30 μM, 2–8 days) decreased ( P < 0.05) AMH mRNA (d4–8) and protein (d6–8). Recombinant AMH (100–400 mg/ml) in PND4 ovaries cultured 8 days ± VCD (30 μM) caused an increase ( P < 0.05) in primordial, and a decrease ( P < 0.05) in small primary follicles, supporting that AMH retarded primordial follicle recruitment. However, no concentration of AMH had an effect on VCD-induced ovotoxicity. Whereas, VCD caused a reduction in expression of AMH (d4–d8), it followed previously reported initial disruptions in Kit signaling induced by VCD (d2). Thus, collectively, these results do not support a mechanism whereby VCD causes ovotoxicity via generalized activation of primordial follicle recruitment, but instead provide further support for the specificity of other intracellular mechanisms involved in VCD-induced ovotoxicity.