The aromatase-knockout (ArKO) mouse provides a useful model to examine the role that estrogens play in development and homeostasis in mammals. Lacking a functional Cyp19 gene, which encodes aromatase, the ArKO mouse cannot synthesize endogenous estrogens. We examined the adipose depots of male and female ArKO mice, observing that these animals progressively accumulate significantly more intraabdominal adipose tissue than their wild-type (WT) littermates, reflected in increased adipocyte volume at gonadal and infrarenal sites. This increased adiposity was not due to hyperphagia or reduced resting energy expenditure, but was associated with reduced spontaneous physical activity levels, reduced glucose oxidation, and a decrease in lean body mass. Elevated circulating levels of leptin and cholesterol were present in 1-year-old ArKO mice compared with WT controls, as were elevated insulin levels, although blood glucose levels were unchanged. Associated with these changes, a striking accumulation of lipid droplets was observed in the livers of ArKO animals. Our findings demonstrate an important role for estrogen in the maintenance of lipid homeostasis in both males and females.estrogen deficiency ͉ obesity ͉ insulin ͉ cholesterol ͉ leptin A romatase is encoded by the Cyp19 gene and catalyzes the final step in the biosynthesis of C 18 estrogens from C 19 steroids. The sexually dimorphic distribution of adipose tissue in humans has implicated sex steroids in the regulation of adiposity and distribution of fat depots. Thus, whereas premenopausal women tend to have a lower body or gynoid distribution of fat, men and postmenopausal women tend to have an upper body or android distribution of fat. This phenotype is associated with a greater risk of insulinresistant diabetes, cardiovascular disease, and breast cancer (1). Estrogen insufficiency is thought to be largely responsible for the increase in adiposity during menopause because postmenopausal women who receive estrogen replacement therapy do not display the characteristic abdominal weight gain pattern usually associated with menopause (2). The role that estrogens play in lipid metabolism in the body is also highlighted by the fact that individuals of both sexes with natural mutations of the gene encoding aromatase, the enzyme responsible for estrogen biosynthesis, develop truncal obesity, insulin resistance, hypercholesterolemia, and hypertriglyceridemia (3-6).We have recently developed a mouse model of estrogen insufficiency by targeted disruption of the aromatase gene: the aromatase-knockout (ArKO) mouse (7). In the course of these studies, we observed that the animals displayed a progressive increase in adiposity as compared with wild-type (WT) littermates. The aim of the present investigation was to characterize the obese phenotype of these animals in the expectation that this would throw light on the role of estrogens in lipid homeostasis. Materials and MethodsMice. ArKO mice were generated by disrupting the Cyp19 gene as described (7). Heterozygous males and fema...

Accurate estimation of the number of ovarian follicles at various stages of development is an important indicator of the process of folliculogenesis in relation to the endocrine signals and paracrine/autocrine mechanisms that control the growth and maturation of the oocytes and their supporting follicular cells. There are 10-fold or greater differences in follicular numbers per ovary at similar ages and/or strains reported in earlier studies using various methods, leading to difficulties with interpretation of ovarian function in control vs experimental conditions. This study describes unbiased, assumption-free stereological methods for quantification of early and growing follicular numbers in the mouse ovary. A fractionator approach was used to sample a defined fraction of histological sections of adult wild-type ovaries. Primordial and primary follicles were counted independently with the optical and physical disector methods. The fractionator/disector methods, which are independent of follicular size or shape, gave estimations of 1930 6 286 (S.E.M.) and 2227 6 101 primordial follicles, and 137 6 25 and 265 6 32 primary follicles per ovary at 70 and 100 days of age respectively. From exact counts on serial sections, secondary and later follicular numbers at 100 days of age were estimated at 135 per ovary. Remnants of zona pellucidae (a marker of previous follicular atresia) were estimated using a fractionator/physical disector approach and were approximately 500 per ovary. The application of the quantitative methods described will facilitate an improved understanding of follicular dynamics and the factors that mediate their growth and maturation and allow for a better comparison between different studies.

Proteins of the Bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult. The recently isolated bcl-w gene encodes a pro-survival member of the Bcl-2 family, which is widely expressed. To explore its physiological role, we have inactivated the bcl-w gene in the mouse by homologous recombination. Mice that lack Bcl-w were viable, healthy, and normal in appearance. Most tissues exhibited typical histology, and hematopoiesis was unaffected, presumably due to redundant function with other pro-survival family members. Although female reproductive function was normal, the males were infertile. The testes developed normally, and the initial, prepubertal wave of spermatogenesis was largely unaffected. The seminiferous tubules of adult males, however, were disorganized, contained numerous apoptotic cells, and produced no mature sperm. Both Sertoli cells and germ cells of all types were reduced in number, the most mature germ cells being the most severely depleted. The bcl-w ؊͞؊ mouse provides a unique model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.

Epidemiologic studies have linked intrauterine growth restriction (IUGR) with an increased incidence of cardiovascular disease later in life; reduced cardiomyocyte number in IUGR hearts may underlie such prenatal programming. Our aim was to examine the effect of IUGR, as a result of maternal protein restriction, on the number of cardiomyocytes in the rat heart at birth. Rats were fed either a low-protein diet (LPD) or a normal-protein diet (NPD) during pregnancy. At birth, the offspring were killed and the hearts were immersion-fixed. The number of cardiomyocyte nuclei in the hearts were stereologically determined using an optical disector-fractionator approach. In some litters, cardiomyocytes were enzymatically isolated from freshly excised hearts and the proportion of binucleated cells was determined. Taking into account the number of binucleated cells, the nuclear counts were adjusted to estimate total cardiomyocyte number. Birth weight and heart weight were significantly reduced in the LPD offspring. This was accompanied by a significant reduction in the number of cardiomyocytes per heart in the LPD offspring compared with the NPD offspring (1.18 Ϯ 0.05 ϫ 10 7 and 1.41 Ϯ 0.06 ϫ 10 7 , respectively; p ϭ 0.001). The number of binucleated cardiomyocytes was low (~3%) and equal in both groups. In conclusion, IUGR as a result of maternal protein restriction leads to a reduction in the number of cardiomyocytes per heart. As cardiomyocyte proliferation is rare after birth, it is plausible that this reduction in cardiomyocytes may lead to compromised cardiac function later in life. Epidemiologic studies have shown a link between low birth weight, as a result of intrauterine growth restriction (IUGR), and an increased incidence of cardiovascular disease later in life (1), suggesting that maternal nutrition may affect the long-term disease profile of offspring. IUGR can result from a lack of nutrients, oxygen, or blood supply to the fetus (2). The link to cardiovascular disease later in life in IUGR infants may relate to underdevelopment of vital organs in utero. Indeed, early studies report that a reduced supply of nutrients during early life, prenatal and postnatal, interferes with the rate of cell multiplication in various organs (3) and that the effect is proportionally more deleterious in tissues with a faster rate of cell multiplication (4). Under these circumstances, growth of the brain is generally "spared" by preferential diversion of blood flow to the brain, whereas growth of other organs is usually proportional to body weight (5). For example, a reduced kidney weight in IUGR rats was shown to be associated with decreased nephron endowment (6,7). The effects of IUGR on the heart are less well defined. In IUGR rats that are exposed to maternal protein restriction, a reduced heart weight is often found (8,9). Alternatively, an increased heart weight as a result of a low-protein diet (LPD) has also been documented (10). Whether IUGR influences the number of cardiomyocytes in the heart is still unclear. If IUGR ...

CENP-B is a constitutive centromere DNA-binding protein that is conserved in a number of mammalian species and in yeast. Despite this conservation, earlier cytological and indirect experimental studies have provided conflicting evidence concerning the role of this protein in mitosis. The requirement of this protein in meiosis has also not previously been described. To resolve these uncertainties, we used targeted disruption of the Cenpb gene in mouse to study the functional significance of this protein in mitosis and meiosis. Male and female Cenpb null mice have normal body weights at birth and at weaning, but these subsequently lag behind those of the heterozygous and wild-type animals. The weight and sperm content of the testes of Cenpb null mice are also significantly decreased. Otherwise, the animals appear developmentally and reproductively normal. Cytogenetic fluorescence-activated cell sorting and histological analyses of somatic and germline tissues revealed no abnormality. These results indicate that Cenpb is not essential for mitosis or meiosis, although the observed weight reduction raises the possibility that Cenpb deficiency may subtly affect some aspects of centromere assembly and function, and result in reduced rate of cell cycle progression, efficiency of microtubule capture, and/or chromosome movement. A model for a functional redundancy of this protein is presented.

Testosterone (T) has been shown to be essential for the completion of spermiogenesis. Our previous studies showed that when intratesticular T was suppressed for 11 wk, the conversion of round spermatids between stages VII and VIII of the spermatogenic cycle was markedly suppressed and that elongated spermatids were undetectable. The fate of the round spermatids that did not proceed through this conversion was unclear. The current study aimed to investigate this T-dependent loss of round spermatids during stages VII and VIII. Adult male Sprague-Dawley rats received 24-cm T implants for 1 wk to suppress LH while maintaining spermatogenesis. The T24 implants were removed and replaced with 3-cm T plus 0.4-cm estradiol (TE treatment) to suppress intratesticular T and spermatogenesis, and animals were killed at 0 and 4 days and 1, 2, 3, 4, and 6 wk later. The number of homogenization-resistant elongated spermatids in the testis was counted, and round spermatid populations in stages VII and VIII were quantified using stereological techniques. The hourly production rates (HPR) were calculated, and a ratio was made between the HPR of round spermatids in stages VII and VIII to assess the efficiency of their conversion through these stages. Testicular T levels were suppressed to 2-4% of control values by TE treatment. After 2 wk of TE treatment, the number of homogenization-resistant elongated spermatids was significantly suppressed, falling to < 0.5% of the control value by 6 wk. The HPR of round spermatids in stages VII and VIII was not affected by up to 2 wk of TE treatment, nor was the conversion between these stages interrupted. After 3 wk of TE treatment, the HPR of round spermatids in stages VII and VIII was significantly suppressed, as was the conversion between these stages, the ratio falling to 27% of the control value by 6 wk. In rats treated with TE, histological examination of the cauda epididymidis showed occasional round spermatids after 3 wk of treatment, and large numbers after 6 wk. We conclude that the failure of round spermatids to complete spermiogenesis following T withdrawal is due to stage-specific detachment of round spermatids between stages VII and VIII.

The historical background to contemporary approaches to the estimation of cell/nuclear number and volume in the testes is reviewed. The limitations of older geometric model-based approaches to the estimation of cell/nuclear number are discussed, and the need for absolute estimates of cell number rather than ratio estimates is examined. The physical and optical disector approaches to the direct estimation of numerical density and, hence, absolute cell number are presented together with data illustrating their operational efficiency in the testis. New approaches to the direct estimation of nuclear/cell volume, using the point-sampled intercept family of methods, are presented and illustrated, using the example of the Sertoli cell nucleus. The use of both classical transverse and the newer vertical section approaches is explored. Estimation of Sertoli cell/nuclear volume in the volume (point-sampled intercept procedure) and number (nucleator and rotator methods) distributions on both conventional transverse and vertical sections is discussed. The use of transverse sections of the testis is shown to produce a consistent bias in the estimation of Sertoli cell nuclear volume in 120-day-old animals, with all the estimators. Comparison of the Sertoli cell nuclear volume (measured on vertical sections) in the volume and number-weighted distribution suggests a coefficient of variation of volume in the number distribution of 0.4-0.5, suggesting either a random or stage-dependent variation in Sertoli cell nuclear size which requires further exploration.

The influence of the Leydig cells on the leukocyte population of the testis was investigated. Leydig cells were destroyed by ethane dimethane sulfonate (EDS) treatment in adult male rats, with or without low-dose s.c. testosterone implants to prevent Leydig cell recovery. Leukocytes were counted in perfusion-fixed frozen testis sections, by use of cell-specific monoclonal antibodies (mAbs) with immunoperoxidase detection, or toluidine blue staining. The majority (81%) of testicular leukocytes (OX1+) were immunopositive for the resident macrophage-specific mAb, ED2, and/or the monocyte/macrophage/dendritic cell mAb, ED1. The remaining leukocytes were principally T lymphocytes (R73+). B lymphocytes (OX33+) and metachromatic mast cells were not observed in the normal testis. Treatment with EDS caused a transient increase in ED1+, ED2+, and R73+ cell numbers in the testis, although other evidence of an inflammatory reaction, such as increases in major histocompatibility complex class II antigen, interleukin-2 receptor expression, or capillary permeability, were not observed. At 21 days after EDS treatment, there was a significant decline in macrophage numbers (to approximately 50% of control testis), and T lymphocytes returned to pretreatment levels. After Leydig cell recovery (41 days after treatment), macrophages also returned to pretreatment levels in EDS-treated rats, but remained reduced in EDS-treated animals with testosterone implants. In addition, EDS treatment stimulated a progressive increase in intertubular mast cells, which was significantly inhibited in the testosterone-implanted rats. The data indicate that numbers of testicular macrophages and mast cells, but not of lymphocytes, within the adult rat testis are directly or indirectly regulated by the Leydig cells.