15 research outputs found
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