Centromere Protein B Null Mice are Mitotically and Meiotically Normal but Have Lower Body and Testis Weights

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

Quantitative (stereological) study of the normal spermatogenesis in the adult monkey (Macaca fascicularis)

Germ cell and Sertoli cell numbers were estimated in six normal adult monkeys (Macaca fascicularis) using a contemporary unbiased and efficient stereological method--the optical disector. The data was used to assess the efficiency of spermatogenesis from type B spermatogonia to elongated spermatids. Animals underwent orchidectomy, and the right testis (volume 17.5 +/- 1.7 cm3 [mean +/- SEM], range 13.2-25.1 cm3) was fixed in Bouin's fluid. Blocks were embedded in methacrylate resin and germ cells were counted in thick (25 microm) sections using the optical disector in conjunction with a systematic uniform random-sampling protocol. The total numbers of Sertoli cells and all germ cells per testis were 566 +/- 43 (419-683) million and 12.8 +/- 1.6 (9.0-20.2) billion, respectively. On average, one Sertoli cell supported 12.4 +/- 1.9 (range 8.2-18.4) step 1-12 spermatids, 3.1 +/- 0.4 (2.3-4.5) pachytene spermatocytes, and 23.7 +/- 4.1 (15.0-39.0) total germ cells. Sertoli cell number correlated poorly with both testicular size (correlation coefficient r = -0.12) and germ cell numbers (r = -0.35 with total germ cell number). However, testicular size had a consistent and significant correlation with germ cell numbers (r = 0.97 with total germ cell number). The conversion ratio of pachytene spermatocytes to step 1-12 spermatids was 3.94 +/- 0.19, which is close to the theoretical maximum of 4. Similarly, the conversion between other cell types was consistently close to the maximum theoretical value. We conclude that the efficiency of spermatogenesis in the adult monkey is high, with stepwise conversion being consistently close to the maximal values. The capacity of Sertoli cells to support a cohort of germ cells varies widely between monkeys. Although absolute number of cells per testis is always the preferred parameter, it cannot always be obtained in an experimental situation where cost and ethical constraints mean that biopsies, rather than whole testes, are collected. Thus, if absolute data on germ cell numbers are not available, experimental outcomes impacting on cells beyond preleptonene spermatocytes may be best expressed in terms of changes in germ cell conversion rather than the traditional germ cell: Sertoli cell ratio

Immature spermatids are not prevalent in semen from men who are receiving androgen-based contraceptive regimens

OBJECTIVE: To determine whether immature spermatids increase in semen in response to hormonal contraceptive treatments. Such a finding would support the existence of a defect in spermiogenesis, which in turn may explain the reported variability in sperm output. DESIGN: Semen smears were obtained from healthy men undergoing randomized control trials of T plus progestin contraceptive treatments. PATIENT(S): Healthy men (21-49 years) with normal semen analyses received T (50-100 mg IM weekly) in combination with either desogestrel (150-300 microg daily, n = 5) or levonorgestrel (125-250 microg daily, n = 10) for 24 weeks. Semen smears were made during spermatogenic suppression and recovery. Nine control subjects were also assessed. MAIN OUTCOME MEASURE(S): Semen analyses were performed using World Health Organization criteria. Immature spermatids and white blood cells in semen were identified by immunostaining with monoclonal antibodies to the human intra-acrosomal antigen SP-10 and the ubiquitous white cell CD-45 antigen, respectively. RESULT(S): In a total of 14 normal ejaculates (9 control and 5 pretreatment) 74+/-14 million/mL sperm (mean+/-SEM) were seen together with a few immature spermatids (0.69+/-0.20 million/mL). During contraceptive treatments, spermatid number decreased in parallel with the sperm concentration and spermatids disappeared in most subjects. No significant changes were seen in either leukocyte or immunonegative round cell concentration (0.41+/-0.25 and 0.25+/-0.09 million/mL in controls, respectively) in response to treatments. CONCLUSION(S): Spermatid sloughing, as assessed by the ejaculation of immature spermatids, is not a feature of T-induced spermatogenic regression in men; rather, the decline in both mature and immature germ cells in the ejaculate probably results from a decline in the number of precursor cells, ultimately resulting in severe oligo- or azoospermia. Detailed studies on the sites of spermatogenic interruption are required to understand the variability in responses seen after contraceptive therapies in men

Impairment of Spermatogonial Development and Spermiation after Testosterone-Induced Gonadotropin Suppression in Adult Monkeys (Macaca fascicularis)

Human male hormonal contraceptive regimens do not consistently induce azoospermia, and the basis of this variable response is unclear. This study used nine adult macaque monkeys (Macaca fascicularis) given testosterone (T) implants for 20 weeks to study changes in germ cell populations in relation to sperm output. Germ cell numbers were determined using the optical disector stereological method. Four animals achieved consistent azoospermia (azoo group), whereas five animals did not (nonazoo group). T-induced gonadotropin suppression in all animals decreased A pale (Ap) spermatogonia to 45% of baseline within 2 weeks, leading to decreased B spermatogonia (32--38%) and later germ cells (20--30%) after 14 and 20 weeks. Though the reduction in later germ cell types could be primarily attributed to the loss of spermatogonia, the data suggested that some cells were lost during the spermatocyte and spermatid phase of development. B spermatogonial number was more markedly suppressed in azoospermic animals, compared with the nonazoo group, as was the conversion ratio between Ap and B spermatogonia. Abnormal retention of elongated spermatids (failed spermiation) was also prominent in some animals after long-term T administration. We conclude that: 1) the variable suppression of sperm output is attributed to the degree of inhibition of germ cell development from type B spermatogonia onwards, and this is related to the degree of FSH suppression; and 2) inhibition of Ap and B spermatogonial development and of spermiation are the major defects caused by long-term T administration to monkeys

Effect of intrauterine growth restriction on the number of cardiomyocytes in rat hearts

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 ± 0.05 × 10 and 1.41 ± 0.06 × 10, respectively; p = 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. Copyright & 2005 International Pediatric Research Foundation, Inc