Reduction In Number Of Follicles Research Articles

Pcsk6 mutant mice exhibit progressive loss of ovarian function, altered gene expression, and formation of ovarian pathology

Bioactivation of precursor proteins by members of the proprotein convertase (PC) family is essential for normal reproduction. The Pcsk6 gene is a member of the PC family that is expressed in numerous ovarian cell types including granulosa cells and oocytes. We hypothesized that loss of PCSK6 would produce adverse effects in the mouse ovary. Mice incapable of expressing PCSK6 (Pcsk6(tm1Rob)) were obtained, and reproductive parameters (serum hormones, whelping interval, estrus cyclicity, and fertility) were compared to Pcsk6(+/+) mice. While Pcsk6(tm1Rob) female mice are fertile, they manifest reduced reproductive capacity at an accelerated rate relative to Pcsk6(+/+) mice. Reproductive senescence is typically reached by 9 months of age and is correlated with loss of estrus cyclicity, elevated serum FSH levels, and gross alterations in ovarian morphology. A wide range of ovarian morphologies were identified encompassing mild, such as an apparent reduction in follicle number, to moderate--ovarian atrophy with a complete absence of follicles--to severe, manifesting as normal ovarian structures replaced by benign ovarian tumors, including tubulostromal adenomas. Targeted gene expression profiling highlighted changes in RNA expression of molecules involved in processes such as steroidogenesis, gonadotropin signaling, transcriptional regulation, autocrine/paracrine signaling, cholesterol handling, and proprotein bioactivation. These results show that PCSK6 activity plays a role in maintaining normal cellular and tissue homeostasis in the ovary.

The Impact of Dietary Organic and Transgenic Soy on the Reproductive System of Female Adult Rat

The goal of this article was to compare the effects of a prolonged use of organic and transgenic soy on the lipid profile and ovary and uterus morphology. Wistar rats were fed three different diets from weaning until sacrifice at 15 months of age. The three diets were: casein-based diet control group (CG), organic soy-based diet group (OSG), or transgenic soy-based diet group (GMSG). There were no differences in food consumption or in the diet isoflavone components among the groups. Compared with the CG diet, both the OSG and GMSG diets were associated with significant reductions in body weight, serum triglycerides, and cholesterol (P < 0.05) (CG = 406 +/- 23.1; 104.3 +/- 13.2; 119.9 +/- 7.3 GMSG = 368 +/- 17.6; 60.3 +/- 4.6; 83.3 +/- 5.7 OSG = 389 +/- 23.5; 72.3 +/- 12.5; 95.5 +/- 8.0, respectively). The volume density of endometrial glandular epithelium was greater in the GMSG group (29.5 +/- 7.17, P < 0.001) when compared with the CG (18.5 +/- 7.4) and OSG (20.3 +/- 10.6) groups. The length density of endometrial glandular epithelium was shorter in both GMSG (567.6 +/- 41.1) and OSG (514.8 +/- 144.5) diets compared with the CG (P < 0.05) diet. GMSG also resulted in reduced follicle number and increased corpus luteum number compared to the OSG or CG diets (P < 0.05). In summary, both GMSG and OSG diets resulted in decreased body weight and lower serum triglyceride and cholesterol levels, and alterations in uterine and ovarian morphology were also observed. The prolonged use of soy-based diets and their relation to reproductive health warrants further investigation.

Impaired Hair Follicle Morphogenesis and Polarized Keratinocyte Movement upon Conditional Inactivation of Integrin-linked Kinase in the Epidermis

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.

Effect of cadmium on gonadal development in freshwater turtle (Trachemys scripta, Chrysemys picta) embryos

Prior studies on painted turtle (Chrysemys picta) sub-populations near the Massachusetts Military Reservation (MMR), a Superfund site on Cape Cod, Massachusetts, USA, suggest several reproductive deficits which may be related to xenobiotics. Several heavy metals, including cadmium, have been detected in Cape Cod surface water and sediments. The present study was carried out to investigate the effect of an environmentally relevant dose of cadmium on gonadal development during the end of the germ cell migration phase and post-natal gonadal maturation in freshwater turtles. Comparison of cadmium concentration in eggs of C. picta from Cape Cod showed that eggs from the impacted site animals had significantly higher cadmium in yolk than eggs from the reference site animals (7.23 ± 1.95 ng/g vs. 1.31 ± 0.50 ng/g). Gonadal structure and the number of proliferating germ cells of neonates derived from eggs of adult females from these sites showed no marked difference between sites. However, apoptosis of oocytes was significantly increased in neonate C. picta from the impacted pond compared to the reference pond. The effect of an administered environmentally relevant dose of cadmium on germ cell number and oocyte apoptosis was subsequently assessed in lab-reared Trachemys scripta, a closely related freshwater turtle species. Assessment of isotopic cadmium transmission showed that 6.29% of cadmium applied to the eggshell was transmitted through the eggshell to the yolk. The results showed that the total number of germ cells in cadmium-treated (1 μg/g) embryos was less than half that found in control embryos. The reduced germ cell number in Cd-treated embryos suggests that cadmium may reduce proliferation and/or delay migration of germ cells to the genital ridge. The effects of cadmium on turtle gonadal development were found to extend into 3 months post hatch. Proliferation of oocytes was not influenced by exposure to cadmium in ovo. In contrast, apoptosis of oocytes was significantly increased in cadmium exposed T. scripta. Since a lesion at the gonial stages will lead eventually to the depletion of more mature germ cells, the results for neonate turtles suggest that an environmental impact due to a xenobiotic mixture may enhance the rate of apoptosis, thus resulting in the reduction in follicle number seen in adult turtles from the impacted site. Overall, the data provide evidence that environmentally relevant doses of cadmium may affect gonadal developmental processes of freshwater turtles during embryonic and post-natal stages that may result in disruption of reproductive processes later in life.

Localization and developmental expression of the activin signal transduction proteins Smads 2, 3, and 4 in the baboon fetal ovary.

We recently demonstrated that the reduction in the number of primordial follicles in ovaries of near-term baboon fetuses deprived of estrogen in utero was associated with increased expression of alpha-inhibin, but not activin betaA and betaB or the activin receptors. Therefore, we proposed that estrogen regulates fetal ovarian follicular development by controlling the intraovarian inhibin:activin ratio. As a prelude to conducting experiments to test this hypothesis, in the current study we determined whether the primate fetal ovary expressed Smads 2/3 and 4 and whether expression of these activin-signaling proteins was altered in fetal ovaries of baboons in which estrogen production was suppressed. Western blot analyses demonstrated that the 59 kDa Smad 2, 54 kDa Smad 3, and 64 kDa Smad 4 proteins were expressed in fetal ovaries of untreated baboons at both mid and late gestation and that the level of expression was not significantly altered in late gestation by in vivo treatment with CGS 20267 or CGS 20267 and estrogen. Immunocytochemistry localized Smads 2/3 and 4 to cytoplasm of oocytes and pregranulosa cells at midgestation and oocytes and granulosa cells of primordial follicles in late gestation. Smad 4 was also detected in granulosa cell nuclei in late gestation, and nuclear expression appeared to be decreased in fetal ovaries of baboons deprived of estrogen. The site of localization of Smads correlated with localization of the activin receptors IA and IIB, which we previously showed were abundantly expressed in oocytes and (pre)granulosa cells at both mid and late gestation and unaltered by estrogen deprivation. In summary, the results of the current study are the first to show that the intracellular signaling molecules required to transduce an activin signal are expressed in the baboon fetal ovary and that expression was not altered by estrogen deprivation in utero. These findings, coupled with our previous observations showing that estrogen deprivation reduced follicle numbers and upregulated/induced expression of inhibin but not activin or the activin receptors, lend further support to the hypothesis that estrogen regulates fetal ovarian folliculogenesis by controlling the intraovarian activin:inhibin ratio.

Ovarian physiology and age-grading in the rice weevil, Sitophilus oryzae (Coleoptera: Curculionidae)

Physiological and morphological changes in the ovarian system in rice weevils, Sitophilus oryzae (L.) (Coleoptera: Curculionidae), reared on wheat seeds were studied as a function of adult age, mating status, and nutrient availability. X-ray analysis was used to determine time of adult eclosion and the duration of development of pre-emergent weevils within the seeds, a process that lasted almost 4 days at 25°C and 60% r.h. There was no follicular differentiation in pre-emergent weevils. Oocyte maturation began after adults emerged from the seeds and started to feed. There was a significant increase in mean germarium length and the size of proximal follicles within the first 5 days when newly emerged weevils were mated and fed ad lib. Maximum number of follicles and mature eggs per ovariole in mated females occurred at 5–30 days of age. The number of mature eggs decreased in 60-days-old weevils, at the same time that adult mortality increased. Development of the ovarian system was much slower in unmated females than in mated females. Although there was follicle development in unmated females, ovulation never occurred and no eggs were laid. Starvation of mated females resulted in a rapid reduction in numbers of follicles and mature eggs, probably as a result of oosorption. Females were categorized into two nulliparous and three parous stages according to ovarian development and the degree of accumulation of follicular relics. Parity was directly correlated with both weevil age and the number of progeny produced and was the physiological basis used to construct an age-grading model for this species. The method will be useful for determining the age structure and reproductive potential of rice weevil populations in the field.

Responses of seasonally anovulatory mares to daily administration of thyrotropin-releasing hormone and(or) gonadotropin-releasing hormone analog.

Seventeen seasonally anovulatory light horse mares were treated daily, starting January 5 (d 1), for 28 d with GnRH analog (GnRH-A; 50 ng/kg BW) and(or) thyrotropin-releasing hormone (TRH; 5 microg/kg BW) in a 2 x 2 factorial arrangement of treatments to test the hypothesis that combined treatment may stimulate follicular growth and development. Ovaries were examined via ultrasonography and jugular blood samples were collected every 3 d. Frequent blood samples were collected after treatment injections on d 1, 2, 4, 7, 11, 16, and 22; on d 29, all mares received an i.v. mixture of GnRH, TRH, sulpiride, and EP51389 (a growth hormone secretagogue) to assess pituitary responsiveness. No consistent effects (P > 0.1) of treatment were observed for plasma LH, FSH, prolactin, or thyroxine concentrations in samples collected every 3 d. The only effect on ovarian follicle numbers was a reduction in number of follicles 11 to 19 mm in diameter due to TRH treatment (P = 0.029). No mare ovulated during treatment. On the days of frequent sampling, mean LH (P = 0.0001) and FSH (P = 0.001) concentrations were higher in mares receiving GnRH-A and tended to increase from d 1 through 7. In contrast, mean prolactin (P = 0.001) and thyroid-stimulating hormone (P = 0.0001) concentrations were high in mares receiving TRH on d 1 but rapidly decreased thereafter. When mares were administered the secretagogue mixture on d 29, the LH response was greater (P = 0.0002) in mares that had previously received GnRH-A but the FSH response was not affected (P > 0.1); the prolactin response was greater (P = 0.014) and the TSH response was smaller (P = 0.0005) in mares that had previously received TRH. Surprisingly, an immediate growth hormone response to EP51389 was absent in all mares. In conclusion, daily GnRH-A treatment stimulated plasma LH and FSH concentrations immediately after injection; although no long-term elevation in preinjection concentrations was achieved, the responses gradually increased over time, indicating a stimulation of gonadotropin production and storage. Daily treatment with TRH stimulated plasma TSH and prolactin concentrations, but the response diminished rapidly and was minimal within a few days, indicating a depletion of pituitary stores and little or no stimulation of production. There was no beneficial effect of adding TRH treatment to the daily GnRH-A regimen.

Influences of age and ovarian follicular reserve on estrous cycle patterns, ovulation, and hormone secretion in the Long-Evans rat.

This study examined the influences of aging and reduced ovarian follicular reserve on estrous cyclicity, estradiol (E(2)) production, and gonadotropin secretion. Young virgin and middle-aged (MA) retired breeder female rats were unilaterally ovariectomized (ULO) or sham operated (control). Unilateral ovariectomy of young rats reduced the ovarian follicular reserve by one-half, to a level similar to that found in MA controls. Unilateral ovariectomy of MA females reduced the follicular pool further, to one half of MA controls. The incidence of regular cyclicity was significantly lower in MA ULO females than in young controls, with intermediate cycle frequency in young ULO and MA controls. Among cyclic rats, the magnitude of the proestrous LH surge was highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. Similarly, ovulation rates were highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. While young ULO rats exhibited augmented secondary FSH surges on estrous morning, middle-aged ULO females displayed secondary FSH levels comparable to young controls. The effects of age and reduced follicle number on estrous cyclicity and gonadotropin secretion were not due to altered E(2) secretion, as preovulatory E(2) levels were similar among all groups. Thus, experimental reduction in the follicular reserve exerts acute effects on the preovulatory LH surge, ovulation rate, and estrous cyclicity in both young and MA rats. However, decreased follicle number increases FSH levels only in young rats, indicating aging-related alterations in the feedback regulation of FSH.

Cryopreservation of Mouse Ovarian Tissue Following Prolonged Exposure to an Ischemic Environment

In cases in which ovarian tissue is to be cryopreserved for tissue or gene banking it is important to maintain its integrity and viability. This study examined how delays between the death of an animal and the collection/cryopreservation of its ovarian tissue influenced follicle viability. Mouse ovaries were placed in PBS+antibiotic (in vitro) or left within the body (in situ) at room temperature for 0, 3, 6, 12, or 24 h following the death of the donor. These ovaries were cryopreserved at 1°C/min on dry ice or in a −84°C freezer using a passive cooling device or by conventional slow cooling (0.3°C/min). The ovaries were grafted under the kidney capsule of ovariectomized recipient mice and collected 2 weeks later, and the size and number of follicles were determined. Cryopreserved ovarian tissue grafted immediately after the death of the donor contained numerous viable and healthy follicles independent of the cooling procedure (dry ice, 134 ± 32; −84°C, 165 ± 54; slow, 214 ± 55 follicles per half ovary). Tissues stored in vitro before cryopreservation retained viable follicles up to 12 h after death (dry ice, 30 ± 15; −84°C, 86 ± 45; slow, 93 ± 33), whereas tissue left in situ had significantly reduced follicle numbers within 3 h of death (dry ice, 36 ± 12; −84°C, 19 ± 6; slow, 28 ± 7). No significant difference was found between the cooling rates tested, indicating that a passive cooling container which cools at 1°C/min is a suitable alternative to conventional slow cooling. We conclude that ovarian tissues for cryobanking should be cryopreserved as soon as possible after collection or death of the animal to ensure maximal follicular survival.

The role of intra-ovarian interactions in the regulation of follicle dominance.

The processes that precisely control the selection of ovulatory follicles from a growing cohort are poorly understood. This reduction in follicle number occurs through several phases of selection, consequently we limit the use of the term 'selection' to the first major reduction of growing follicles, at the pre- to early antral stage. The final process of selection, achieving the appropriate ovulatory number, is referred to as 'dominance'. We discuss possible mechanisms that could bring about these reductions and highlight intra-ovarian involvement, particularly via follicle-follicle interactions. Analogies are drawn between local ovarian events and processes commonly reported in the determination of cell fate in developmental biology. Two facets of intra-follicular interactions are proposed: initially that follicle-follicle interactions mediate early selection processes at the preantral stage, and later that during antral development dominant follicles directly affect the fate of the subordinate cohort members.

Diminished α-inhibin messenger ribonucleic acid in in vitro fertilization-embryo transfer poor responders reflects declining follicle reserve

To quantitate and compare granulosa cell alpha-inhibin messenger RNA (mRNA) levels in IVF-ET poor and good responders and thereby learn how alpha-inhibin mRNA levels change in states of diminished ovarian responsiveness. Ribonucleic acid analysis of stored luteinized granulosa cell samples. Academic tertiary care institution. Fifty-three women undergoing follicle aspiration for IVF-ET were studied. Patients were classified as poor responders (n = 16) or good responders (n = 37) according to their E2 concentration on the day of hCG; the E2 of poor responders was < 1,000 pg/mL (3,671 pmol/L) and that of good responders was > or = 1,000 pg/mL (3,671 pmol/L). Messenger RNA levels were measured using dot blot RNA analysis. The following parameters were determined or derived: total mRNA levels, total alpha-inhibin mRNA levels, alpha-inhibin mRNA per follicle, and proportional alpha-inhibin mRNA as the ratio of alpha-inhibin mRNA:total mRNA. Proportional alpha-inhibin mRNA and alpha-inhibin mRNA per follicle were not significantly different between poor responders and good responders. Total mRNA and total alpha-inhibin mRNA levels, however, were diminished significantly in poor responders. The observations that proportional alpha-inhibin mRNA and alpha-inhibin mRNA per follicle do not significantly change in poor responders, whereas total alpha-inhibin mRNA does, indicate that the decrease in total alpha-inhibin mRNA in poor responders reflects a decreased pool of total mRNA, likely because of a reduction in follicle number. These findings are in contrast to other recent reports that describe a change in granulosa cell function accompanying states of decreased ovarian responsiveness.

Endocrine aspects of menopause.

Oocyte depletion and ovarian aging results in profound alterations at the biological level. The reduction in numbers of follicles leads to reduction of circulating inhibin and increased serum FSH, characteristic marker of ovarian failure. The remaining follicles are overstimulated and premature ovulation may ensue. This, leads to luteal deficiency and reduction in progesterone production and relative hyperestrogenemia. The above changes characterize the premenopausal period. In a second phase, anovulatory cycles interchange with premature and occasionally normal ovulatory cycles. This phase is characterized by increased FSH and LH and decreased progesterone/estradiol ratio resulting in irregular bleeding, endometrial hyperplasia, polyps, fibromas, mammary dystrophies, disturbance of mood, appetite and thermoregulation. Lastly, the sensitivity of ovarian follicles to FSH and LH is lost with a decline of E2 below 20 pg/ml produced almost exclusively from peripheral conversion from circulating androgens. The beneficial effects of E2 are lost resulting in atrophy of the sensitive tissues, decreased calcium absorption, increased bone resorption, accelerated bone loss and osteoporosis, rise in serum triglycerides, increased VLDL and LDL lipoproteins, increased LDL/HDL cholesterol, a profile which favors atherosclerosis.

Gonadal effects of cancer therapy in girls.

The gonadal histology of 21 prepubertal, intrapubertal, and postpubertal girls who died 1 day to 2 months after cessation of therapy for extragonadal solid tumors was reviewed. In addition to focal or diffuse cortical fibrosis, a reduction in follicle numbers and impaired follicular maturation were observed in cancer patients independent of their pubertal age. These changes appeared to be more severe in malnourished patients and in girls who received multiple agent chemotherapy, with or without irradiation. Both before and after the age of 10 years, most cancer patients had a total number of follicles similar to that of age- and nutrition-matched controls. However, the majority of these girls displayed impaired follicular maturation as demonstrated by reduced numbers of growing and antral follicles compared to controls. Histologic evidence of ovarian damage suggests that the future reproductive performance may be impaired in some female cancer patients treated even before puberty.