Ovarian Defects Research Articles

A 9-Year-Old Girl Presenting Central Precocious Puberty with Polycystic Ovary Syndrome.

A 9-year-old girl presented central precocious puberty (CPP) with polycystic ovary syndrome (PCOS) simultaneously. She had obesity, hirsutism and acanthosis nigricans. Laboratory examination revealed high levels of serum LH, FSH (LH > FSH), testosterone and insulin. The response of dehydroepiandrosterone sulfate after administration of ACTH was normal for a female. She has been treated with GnRH analog and weight reduction. Excessive androgen production results from ovarian defect and therefore, functional ovarian hyperandrogenism and hyperinsulinemia might be major factor in the pathogenesis. This case is the first report of a PCOS woman demonstrating CPP simultaneously.

Ovary signals for directional pollen tube growth

In angiosperms, the female gametophyte has a secluded life; it is protected by several concentric layers that envelop each other. The embryo sac is surrounded by the nucellus, which in turn is wrapped by the integuments forming the ovule, which is nested in the ovary. These wrappings are not hermetic, but contain little ”gates” the pollen tube must traverse on its way towards the embryo sac. Information is emerging that shows that the ovary and ovule provide signals orienting and directimng the pollen tube on the right course. There are three main bodies of evidence supporting this hypothesis. One relates to developmental changes in the female tissues and how they affect pollen tube growth. The second refers to defective ovule mutants, which induce defective pollen tube guidance. And the third relates to the possible molecules involved in this signalling. Here, information gathered along these three main lines of evidence is reviewed. All converge to the conclusion that different checkpoints exist all along the pollen tube pathway. These checkpoints provide active signalling that guides the pollen tube to its destination, the embryo sac.

Resumption of ovarian function during lactational amenorrhoea in breastfeeding women with polycystic ovarian syndrome: endocrine aspects.

The aim of this study was to evaluate the changes in gonadotrophin concentrations and the dynamics of the episodic fluctuations of circulating LH during night-time, in fully breastfeeding normal women and in those with polycystic ovarian syndrome (PCOS) during lactational amenorrhoea and after weaning, in order to provide insights into the onset of this syndrome. Additionally, ovarian activity was evaluated by ultrasound examination and steroid concentrations. Twelve lactating PCOS (LPCOS) women and six normal lactating (NL) women of similar age were selected. On the 4th and 8th week postpartum (PP) and eight weeks after weaning, blood samples were collected every 10 min (10.00--20.00h). Gonadotrophin concentrations were determined in all samples. Steroid hormones were measured in one fasting sample and ovarian morphology was assessed by ultrasound. On the 8th week PP, LH pulse frequency was higher and FSH concentrations were lower in LPCOS women compared with NL women, and steroid hormone concentrations remained low, except for androstenedione which was higher in LPCOS patients. After weaning, similar differences were observed between both groups. PCOS patients also showed enlarged ovaries with a PCOS pattern in the three study periods. The enlarged ovaries associated with higher androstenedione concentrations suggest that PCOS is a primary ovarian defect, making it difficult to establish if the abnormal LH pattern observed in these women is primary or secondary to the ovarian dysfunction.

Targeted disruption of the aromatase P450 gene (Cyp19) in mice and their ovarian and uterine responses to 17beta-oestradiol.

Aromatase P450 (CYP19) is an enzyme catalysing the conversion of androgens into oestrogens. We generated mice lacking aromatase activity (ArKO) by targeted disruption of Cyp19 and report the characteristic features of the ArKO ovaries and uteri as revealed by histological and biochemical analyses. ArKO females were totally infertile but there were as many developing follicles in their ovaries at 8 weeks of age as in wild-type ovaries. Nevertheless, no typical corpus luteum was observed in the ArKO ovaries. Electron microscopy revealed the presence of well-developed smooth endoplasmic reticulum, few lipid droplets and mitochondria with less organized tubular structures in the ArKO luteinized interstitial cells. These ultrastructural features were different from those of the wild-type interstitial cells, where there are many lipid droplets and mitochondria with well-developed tubular structures, characteristic of steroid-producing cells. When ArKO mice were supplemented with 17beta-oestradiol (E(2); 15 microg/mouse) every fourth day from 4 weeks of age for 1 month, increased numbers of follicles were observed in the ovaries as compared with those of untreated ArKO mice, although no typical corpus luteum was detectable. Ultrastructural analysis revealed the disappearance of the accumulated smooth endoplasmic reticulum in the luteinized interstitial cells after E(2 )supplementation. Transcripts of pro-apoptotic genes such as p53 and Bax genes were markedly elevated in the ArKO ovaries as compared with those of wild-type mice. Although E(2) supplementation did not cause suppression of the elevated expression of p53 and Bax mRNAs, it caused marked enhancement of expression levels of lactoferrin and progesterone receptor mRNAs in the uteri as well as increases in uterine wet weight. At 8 months of age, ArKO mice developed haemorrhages in the ovaries, in which follicles were nearly depleted, while age-matched wild-type females still had many ovarian follicles. Furthermore, macrophage-like cells were occasionally observed in the ArKO ovarian follicles. These results suggested that targeted disruption of Cyp19 caused anovulation and precocious depletion of ovarian follicles. Additionally, analysis of mice supplemented with E(2) demonstrated that E(2) apparently supports development of ovarian follicles, although it did not restore the defect in ovulation.

Association between Estrogen Receptor-β Gene Polymorphisms and Ovulatory Dysfunctions in Patients with Menstrual Disorders

Estrogen plays a significant role in human ovulation. It acts as an important positive regulator of the preovulatory gonadotropin surge necessary to initiate the cascade of events leading to ovulation. The steroid hormone exerts its physiological responses through the estrogen receptor (ER), of which two subtypes, ERalpha and ERbeta, are known. ERbeta messenger ribonucleic acid occurs maximally in the ovaries and granulosa cells; thus, ERbeta may be essential for normal ovulation. In a recent gene knockout study, it has been shown that ERbeta gene null female mice develop normal reproductive tract and ovaries during pre- and neonatal periods, but have an abnormal frequency of spontaneous ovulation in adulthood. In the present case-control study, we explored the association of two recently described ERbeta gene polymorphisms, RSAI and ALUI, with ovulatory dysfunctions. The respective frequencies of these polymorphisms were significantly higher in patients than in controls (P= 0.009 and P= 0.059). The polymorphisms were significantly associated with ovulatory dysfunctions, especially in patients homozygous for the polymorphisms (P = 0.016 and P = 0.038, respectively). The compound homozygosity of the polymorphisms was seen only in patients (n = 5) and not controls (P = 0.009). The serum levels of LH, FSH, and progesterone were lower in the homozygous and compound homozygous than in the respective nonpolymorphic patients. All five compound homozygous patients had ovulatory dysfunctions with no etiological pathology. Our results suggest that ERbeta gene RSA:I and ALU:I polymorphisms may be associated with ovulatory defects in some patients, especially those with unknown causes.

Estrogen deficiency, obesity, and skeletal abnormalities in follicle-stimulating hormone receptor knockout (FORKO) female mice.

Targeted disruption of the receptor for glycoprotein hormone, FSH (FSH-R) causes a gene dose-related endocrine and gametogenic abnormality in female mice. The resulting FSH-R knockout (FORKO) mutants have disordered estrous cycles, ovulatory defects, and atrophic uterus. The heterozygous animals that initially show reduced fertility undergo early reproductive senescence and stop breeding altogether. Lack of FSH-R signaling in females causes severe ovarian underdevelopment producing chronic estrogen deficiency. This was accompanied by increases in serum testosterone levels. Ovarian aromatase gene transcription and translation are unaltered in the mutants. Early loss of estrogen in the null mutants leads to obesity and skeletal abnormalities that intensify with age producing (kyphosis), a hunchback appearance. Both these changes also become apparent in older heterozygous mice coincident with early reproductive senescence. The expression of nuclear estrogen receptor(s) alpha and beta genes and the corresponding proteins in the ovary and uterus of FORKO mice appear to be intact. The loss of ovarian estrogen creates an imbalance in A and B forms of the progesterone receptor in the uterus of both heterozygotes and null mutants. Some of the changes we have documented here in FORKO mice are reminiscent of the ovarian dysfunction and other major symptoms that are usually associated with estrogen deficiency. In null mutants, estradiol-17beta administration promptly induced uterine growth and reversed the accumulation of adipose tissue indicating that estrogen receptors are functional. Thus, the phenotypes evident in these genetically altered FSH-R mutants may provide an experimental system to explore the effects of estrogenic compounds on different targets including the ovary in a nonsurgical setting.

Estrogen Deficiency, Obesity, and Skeletal Abnormalities in Follicle-Stimulating Hormone Receptor Knockout (FORKO) Female Mice

Targeted disruption of the receptor for glycoprotein hormone, FSH (FSH-R) causes a gene dose-related endocrine and gametogenic abnormality in female mice. The resulting FSH-R knockout (FORKO) mutants have disordered estrous cycles, ovulatory defects, and atrophic uterus. The heterozygous animals that initially show reduced fertility undergo early reproductive senescence and stop breeding altogether. Lack of FSH-R signaling in females causes severe ovarian underdevelopment producing chronic estrogen deficiency. This was accompanied by increases in serum testosterone levels. Ovarian aromatase gene transcription and translation are unaltered in the mutants. Early loss of estrogen in the null mutants leads to obesity and skeletal abnormalities that intensify with age producing (kyphosis), a hunchback appearance. Both these changes also become apparent in older heterozygous mice coincident with early reproductive senescence. The expression of nuclear estrogen receptor(s) α and β genes and the corresponding proteins in the ovary and uterus of FORKO mice appear to be intact. The loss of ovarian estrogen creates an imbalance in A and B forms of the progesterone receptor in the uterus of both heterozygotes and null mutants. Some of the changes we have documented here in FORKO mice are reminiscent of the ovarian dysfunction and other major symptoms that are usually associated with estrogen deficiency. In null mutants, estradiol-17β administration promptly induced uterine growth and reversed the accumulation of adipose tissue indicating that estrogen receptors are functional. Thus, the phenotypes evident in these genetically altered FSH-R mutants may provide an experimental system to explore the effects of estrogenic compounds on different targets including the ovary in a nonsurgical setting.

Regulation of Cyclooxygenase-2 Induction in the Mouse Uterus During Decidualization: AN EVENT OF EARLY PREGNANCY

The infertility phenotype of cyclooxygenase-2 (Cox-2)-deficient female mice establishes the important role of Cox-2 in pregnancy. Cox-2 deficiency results in defective ovulation, fertilization, implantation, and decidualization; the latter of which can be restored in part by the prostacyclin analog carbaprostacyclin. Uterine Cox-2 expression during early pregnancy shows distinct localization and kinetics in the uterine luminal epithelium and underlying stromal cells, suggesting that expression is tightly regulated. Several intracellular signaling cascades including ERK, p38, and JNK are implicated in vitro as critical components of regulated Cox-2 expression in response to mitogens, growth factors, and cytokines. We investigated the involvement of these signaling pathways during Cox-2 induction in vivo by monitoring uterine kinase activity after intraluminal application of a deciduogenic stimulus. Our results show that the ERK and p38 pathways are activated in uterine preparations as early as 5-min post-stimulation. ERK activation was sustained for several hours with a return to baseline levels by 4 h. p38 activation was rapid with a peak at 5-min post-stimulation and returned to near baseline levels after 45 min. Systemic administration of a MEK inhibitor completely inhibited ERK activation, but did not affect early (2 h) luminal epithelial or late (24 h) stromal Cox-2 expression and only modestly affected decidualization. In contrast, administration of a p38 inhibitor modestly inhibited early Cox-2 expression in the luminal epithelium, while dramatically diminishing late stromal expression. In parallel, induced stromal peroxisomal proliferator activated receptor-delta (PPARdelta) expression is blunted by p38 inhibition. p38 inhibition also significantly inhibited decidualization. These results suggest that p38, but not ERK, activation is required for induced Cox-2 and PPARdelta expression during decidualization. In addition, inhibition of p38 led to decreased decidualization suggesting that an intracrine prostanoid pathway consisting of Cox-2, prostacyclin, and PPARdelta is required for maintenance of early pregnancy.

Physiological functions of prostanoid receptors and their subtypes

Prostaglandins exert a wide variety of biological actions through specific receptors. The prostaglandins PGE2 and PGF2 alpha have been suggested to affect many physiological processes. There have been four pharmacologically identified receptor subtypes, EP1 through EP4 for PGE2 and a single receptor type, FP for PGF2 alpha. However, it is yet unknown as to which receptor is involved in each process. To make this clear, we investigated the distribution of these receptors in various systems and established mice deficient in each receptor and examined their physiology. Finally, we found that these receptors are involved in several processes associated with reproduction physiology. FP-deficient mice are able to become pregnant, but cannot deliver their pups because of the lack of parturition; luteolysis does not occur normally. EP2-deficient mice are able to become pregnant, but their litter sizes are much reduced, which is due to defects in ovulation and fertilization; cumulus cell function is impaired. EP4-deficient mice die within a few days after birth because of patency of the ductus arteriosus; remodeling of cardiovascular system during birth is impaired. Thus, the E- and F-types of prostaglandins play roles in cumulus function, luteolysis and ductus closure through EP2, FP and EP4, respectively.

Fertility of Y(TIR).B6 sex-reversal females with XX orthotopic ovarian transplants.

When the Y chromosome of Mus musculus domesticus (Y(TIR)) was introduced onto the C57BL/6J (B6) mouse background, testis development was impaired and half of the XY progeny (Y(TIR).B6) developed a female phenotype. Y(TIR).B6 fetal ovaries showed massive death of medullary oocytes and, after birth, produced abnormal levels of steroid hormones, exhibited irregular estrous cycles, and failed to become fertile. In this study we examined whether alterations during perinatal development observed in Y(TIR).B6 ovaries permanently impaired the establishment of the hypothalamus-pituitary-ovary axis (HPOa). B6 fetal and postnatal ovaries at different stages (fetal, infantile, or adult) were transplanted orthotopically (to the ovarian bursa) to either ovariectomized B6 normal females or Y(TIR).B6 sex-reversal females. Percentage of pregnancy, litter size, and capacity to feed pups were recorded. Reciprocally, XY(TIR).B6 ovaries were orthotopically transplanted into B6 females. After crossing with fertile males, several Y(TIR).B6 sex-reversal females with B6 ovarian transplants at all ages became pregnant, had offspring, and fed their pups. On the other hand, none of the B6 female hosts with XY(TIR) ovaries became pregnant. Results demonstrated that Y(TIR).B6 sex-reversal females maintain a functional HPOa and that their failure to reproduce is primarily due to an ovarian defect.

Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E2 and interleukin-1beta.

Mice carrying a null mutation for either of the two cyclooxygenase (COX) isoenzymes, necessary for prostanoid production, exhibit several isotype-specific reproductive abnormalities. Mice deficient in COX-1 are fertile but have decreased pup viability, whereas mice deficient in COX-2 fail to ovulate and have abnormal implantation and decidualization responses. The present study identifies the specific contribution of each COX isoenzyme in hypothalamic, pituitary, and ovarian function and establishes the pathology and rescue of the anovulatory syndrome in the COX-2-deficient mouse. In both COX-1- and COX-2-deficient mice, pituitary gonadotropins were selectively increased, whereas hypothalamic LHRH and serum gonadotropin levels were similar to those in wild-type animals (+/+). No significant differences in serum estrogen or progesterone were noted among the three genotypes. Exogenous gonadotropin stimulation with PMSG and hCG produced a comparable 4-fold increase in ovarian PGE2 levels in wild-type and COX-1(-/-) mice. COX-2(-/-) mice had no increase in PGE2 over PMSG-stimulated levels. Wild-type and COX-1(-/-) mice ovulated in response to PMSG/hCG; very few COX-2(-/-) animals responded to this regimen. The defect in ovulation in COX-2 mutants was attributed to both an abnormal cumulus oophorum expansion and subsequent stigmata formation. Gonadotropin stimulation and concurrent treatment with PGE2 or interleukin-1beta resulted in ovulation of COX-2(-/-) mice comparable to that in COX-2(+/+), whereas treatment with PGF2alpha was less effective. Collectively, these data demonstrate that COX-2, but not COX-1, is required for the gonadotropin induction of ovarian PG levels; that COX-2-related prostanoids are required for stabilization of the cumulus oophorum during ovulation; and that ovulation can be restored in the COX-2(-/-) animals by simultaneous treatment with gonadotropins and PGE2 or interleukin-1beta.

Adjunctive medical treatments in follicular stimulation

Approximately 25% to 35% of couples that seek infertility services suffer from defects in ovulation.This diagnosis represents the single leading cause of infertility disorders, and makes medical ovulation induction the most common intervention for the treatment of infertility. There are two classes of correctable ovulation defects, and correct classification is correlated with treatment and prognosis. World Health Organization (WHO) I patients have ovulation defects associated with low estrogen and low gonadotropin levels and fail to exhibit withdrawal bleeding after progestin challenge. WHO II ovulation disorders occur in estrogenized/androgenized women who, in general, menstruate. Other names for these conditions include hypogonadotropic hypogonadism (WHO I) and polycystic ovarian disease (WHO II).

7 The use of gonadotrophin-releasing hormone antagonists in polycystic ovarian disease

Polycystic ovarian disease (PCOD) is characterized by anovulation, eventually high luteinizing hormone (LH) levels, with increased LH pulse frequency, and hyperandrogenism. As the aetiology of the disease is still unknown, gonadotrophin-releasing hormone (GnRH) antagonists, competitive inhibitors of GnRH for its receptor, are interesting tools in order to study and treat the role of increased LH levels and pulse frequency in this disease. Their administration provokes a rapid decrease in bioactive and immunoactive LH followed by a slower decrease in follicle-stimulating hormone (FSH). In patients with PCOD, the suppression of gonadotrophin secretion eradicates the symptoms of the disease as long as the treatment lasts. Several authors have suggested that increased plasma LH levels have deleterious effects on the fertility of women with PCOD. Indeed, fewer spontaneous pregnancies with more miscarriages are observed when plasma LH levels are high. Assisted reproduction techniques such as in vitro fertilization (IVF) have provided other clues to the role of the LH secretory pattern in women with PCOD. The number of oocytes retrieved, the fertilization rate and the cleavage rate are lower in PCOD patients undergoing IVF and this is inversely correlated with FSH:LH ratio. These abnormalities are corrected when endogenous secretion of LH is suppressed. On the other hand, implantation and pregnancy rates after IVF are similar to those observed in control women. New GnRH antagonists are devoid of side effects and suppress LH secretion within a few hours without a flare-up effect. This action lasts for 10-100 hours. When GnRH antagonists are associated with i.v. pulsatile GnRH, this combination both suppresses the effect of endogenous GnRH and because of the competition for GnRH receptors restores a normal frequency of LH secretion. We have studied two women with PCOD, administering first 10 mg s.c. every 72 hours for 7 days of the GnRH antagonist Nal-Glu, then adding on top i.v. pulsatile GnRH: 10 micrograms/pulse every 90 minutes for 15 days. We thus succeeded in normalizing LH secretion pattern and observed a significant decline in testosterone levels. We failed to induce appropriate ovarian response and ovulation. In conclusion, the combination of GnRH antagonist and GnRH pulsatile treatment can re-establish normal LH secretory pattern in patients with PCOD. The failure to induce ovulation with this regimen suggests the existence of an inherent ovarian defect in women with PCOD.

SHORT- AND LONG-TERM EFFECTS OF OVULATION INDUCTION

Ovulation induction using clomiphene citrate, gonadotropins, and gonadotropin-releasing hormone is reviewed. The short- and long-term consequences of these therapies are discussed in detail.

The role of nitric oxide in oocyte meiotic maturation and ovulation: meiotic abnormalities of endothelial nitric oxide synthase knock-out mouse oocytes.

Evidence supports the involvement of nitric oxide (NO) in ovulation, steroidogenesis, and atresia-related apoptosis. This study was designed to investigate the role of endothelial nitric oxide synthase (eNOS)-derived NO in ovulation, oocyte meiotic maturation, and ovarian steroidogenesis using wild-type (WT) mice and mice in which the gene for eNOS had been deleted (eNOS knock-out). We observed that mature eNOS knock-out females have significantly fewer pups born in each litter and a higher mortality rate of pups than those born to heterozygote or WT females (P < 0.05). To determine the influence of eNOS deficiency on ovarian function, immature WT and eNOS knock-out mice were superovulated by injecting PMSG (5 IU) followed by an injection of hCG (5 IU, i.p.) 48 h later. To determine whether murine oocytes expressed eNOS before (0 and 8 h post-hCG) and after ovulation (16 h post-hCG). we performed immunofluorescent staining. Positive specific staining for eNOS was observed on the surface of ovarian and ovulated oocytes obtained from WT mice, but not on oocytes from eNOS knock-out mice. To determine the role of eNOS-derived NO in ovulation, ovulated oocytes were counted 16 h post-hCG. eNOS knock-out females showed a significant reduction (by 63%; P < 0.0001) in ovulatory efficiency compared with WT females. The reduction in the ovulation rate in eNOS-deficient mice compared with that in WT mice was also associated with a higher concentration of estradiol (P < 0.01) without significant changes in the plasma progesterone level. eNOS deficiency impaired not only ovulation, but also oocyte meiotic maturation. Ovulated oocytes were classified as being in one of the following stages of meiosis: metaphase I, metaphase II, or showing atypical (degenerative) morphology. We observed that fewer oocytes from eNOS knock-out mice had entered metaphase II of meiosis, and a greater percentage remained in metaphase I or were atypical (P < 0.002) relative to those in WT mice. Furthermore, many oocytes that showed either a delay in meiotic maturation or abnormal morphology were undergoing cell death. Our results support a role for NO in the ovulatory process. The ovarian defects observed in the eNOS knock-out mice suggest that eNOS-derived NO is a modulator of oocyte meiotic maturation.

Hyperandrogenic anovulation (the polycystic ovary syndrome)--back to the ovary?

Hyperandrogenic anovulation is characterized by polycystic appearance of the ovaries, elevated free serum testosterone with decreased concentrations of serum sex hormone binding globulin, an increased ratio of luteinizing hormone to follicle stimulating hormone and varying degrees of insulin resistance. We hypothesize that this is the result of variably increased 'ovarian androgenic insulin responsiveness' acting in combination with body mass. Women who develop hyperandrogenism and anovulation when lean (having normal serum insulin concentrations) represent the more severely affected individuals, whereas those who can resume ovulation by losing weight (and lowering their elevated serum insulin) represent a milder form. The tendency of these women to develop ovarian hyperstimulation syndrome despite hypophyseal desensitization suggests a primary ovarian defect which is apparently more pronounced in lean subjects. The unique ability of surgical damage to the ovary to induce ovulation, raises the possibility that inflammatory-like tissue remodelling has a major role in rescuing follicles from androgen-induced atresia. Approaches that may facilitate the study of this possible mechanism may include examination of in-vitro perfused, post-surgery mammalian ovaries and the elucidation of signal transduction mechanism(s) of insulin in the ovary, with special reference to cells emanating from affected women.

Multiple pituitary and ovarian defects in Krox-24 (NGFI-A, Egr-1)-targeted mice.

The zinc finger transcription factor Krox-24 (NGFI-A, Egr-1) is encoded by an immediate-early serum response gene expressed in various physiological situations and tissues. To investigate its function, we have created a null allele. Mice homozygous for the mutation have a reduced body size, and both males and females are sterile. These phenotypes were related to defects in the anterior pituitary of both sexes and in the ovary. In the pituitary, two cell lineages expressing Krox-24 are differentially affected by the mutation: somatotropes present abnormal cytological features and are reduced in number, consistent with the decreased GH content observed in these animals; in contrast gonadotropes are normal in number, but specifically fail to synthesize the beta-subunit of LH. In the ovary, LH receptor expression is prevented, indicating an involvement of Krox-24 at two levels at least of the pituitary-gonadal axis. Our data, together with the results of a previous report describing another Krox-24 mutant allele, suggest that Krox-24 may have two distinct molecular functions in the anterior pituitary: transcriptional activation of the LHbeta gene in gonadotropes and control of cell proliferation and/or survival in somatotropes by unknown mechanisms.

Ovulation defect and its restoration by bone marrow transplantation in osteopetrotic mutant mice of Mitf(mi)/Mitf(mi) genotype.

Mutation within the Mitf gene causes, in microphthalmic Mitf(mi)/Mitf(mi) (mi/mi) mice, multiple defects, including white coat color and functional defects in macrophages and osteoclasts. Our previous mating experiments have demonstrated that the mi mutation reduces the numbers of newborns and induces uterine inversion at delivery. The present study was designed to determine the causes of these pregnancy defects. The histology and number of F4/80-positive macrophages were not different between the ovaries of 23-day-old mi/mi and +/+ mice given eCG 48 h earlier. When ovulation was induced in these mice by hCG, the number of ovulated ova was significantly smaller in mi/mi mice than in wild-type (+/+) mice (p < 0.05). When bone marrow cells from +/+ mice were transplanted i.p. into 42 mi/mi female newborns, successful transplantation was observed in 16 of them at 20 days after birth. In one of these, the upper incisors had erupted. The mean number of tubal ova in mi/mi mice significantly increased after transplantation (p < 0.05) and was almost equal to that of +/+ mice. No uterine inversion occurred at 6 deliveries in 5 mi/mi females after bone marrow transplantation, while it occurred at 4 of 5 deliveries in mi/mi females during the same observation period (p < 0.05). These results indicate that bone marrow-derived cells, defective in mi/mi mice, are necessary for normal ovulation and delivery; the findings are consistent with the notion that macrophages play major roles in ovulation.

The POU Geneceh-18Promotes Gonadal Sheath Cell Differentiation and Function Required for Meiotic Maturation and Ovulation inCaenorhabditis elegans

InCaenorhabditis elegans,specialized contractile myoepithelial cells of the somatic gonad, the gonadal sheath cells, are closely apposed to oocytes and are required for normal meiotic maturation and ovulation. Previously we found that mutations in theceh-18gene, which encodes a POU-class homeoprotein expressed in sheath cells, result in oocyte defects. To determine the basis for these oocyte defects, we have used time-lapse video Nomarski microscopy to observe meiotic maturation, ovulation, and early embryogenesis inceh-18mutants. Inceh-18mutants sheath cell contractions are weaker, less frequent, and uncoordinated throughout the sequence of ovulation events, and ovulation is defective. Defective ovulation can result in the formation of endomitotic oocytes in the gonad, the formation of haploid embryos, and reversals in embryonic polarity.ceh-18mutant oocytes exhibit defects prior to nuclear envelope breakdown, suggesting that they are physiologically different from the wild type. We observed delays in meiotic maturation, as well as maturation out of the normal spatial and temporal sequence, suggesting that proximal sheath cells directly or indirectly promote and spatially restrict meiotic maturation. Analysis of sheath cell differentiation inceh-18mutants using antibodies to proteins of the contractile apparatus reveals that although contractile proteins are expressed, the sheath cells appear disorganized. Transmission electron microscopy reveals thatceh-18mutant sheath cells are morphologically irregular and only loosely cover oocytes. Taken together, these observations indicate thatceh-18is a crucial determinant of sheath cell differentiation, a function required for normal meiotic maturation and ovulation.

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.