Elucidation of the role of LH and FSH during neonatal testicular development and growth in the boar.

The aim was to elucidate the role of LH and FSH in testicular development and growth in the neonatal boar. On Day 10 after birth (Day 0 of study), animals were assigned to one of nine groups (n=6): Group 1, control, no treatment; Group 2, hemicastrated (H); Group 3, H and implanted with GnRH agonist (H+GnRH); Group 4, H+GnRH+FSH 200µg/kg daily from Days 0 to 14 (D0-14); Group 5, H+GnRH+FSH 400µg/kg D0-14; Group 6, H+GnRH+FSH 400µg/kg in PVP D0-14; Group 7, H+GnRH+LH 200µg/kg D0-14; Group 8, H+GnRH+LH 400µg/kg D0-14; Group 9, H+GnRH+LH and FSH 200µg/kg D0-14. The right testis in control and hemicastrated boars was removed on Day 15. Hemicastrated boars had greater (P<0.05) testicular growth than control boars and testicular growth was prevented in boars treated with GnRH agonist. FSH induced Sertoli cell proliferation but not testicular growth whilst LH induced Leydig cell proliferation and testicular growth was similar to control boars but less than hemicastrated boars. LH+FSH induced similar testicular growth as LH alone and neither LH and/or FSH supported testicular hypertrophy in hemicastrated boars. The findings show conclusively for the first time that LH and FSH respectively induce Leydig cell and Sertoli cell proliferation in the neonatal boar. LH additionally supports a normal increase in testicular size in the neonatal boar.

Expression of FGF2 and TGFalpha and testis morphology during testicular hypertrophy subsequent to hemicastration in the neonatal boar.

The objective was to ascertain fibroblast growth factor-2 (FGF2), epidermal growth factor (EGF), and transforming growth factor-alpha (TGFalpha) mRNA expression and testis morphology during accelerated testicular growth after hemicastration in the neonatal boar. On Day 10 after birth (Day 0), boars were assigned to control (n = 28), no treatment; hemicastrated (n = 28), left testis removed. The right testis in both groups (n = 7) was removed on Days 5, 10, 15, and 20. Expression of mRNA for FGF2, EGF, and TGFalpha was determined by qRT-PCR using TaqMan. Testicular morphology was determined on Day 15. On Day 10, hemicastrated boars had a greater (P = 0.01) testis weight (6.2 +/- 0.8 g; mean +/- SEM) than controls (4.3 +/- 0.4 g) and on Day 15 testis weight in hemicastrated boars (8.8 +/- 0.8 g) was twice (P < 0.01) that of control boars (4.2 +/- 0.3 g). Seminiferous tubule volume was approximately doubled in hemicastrated boars (P < 0.01) and was associated with an increase (P < 0.01) in Sertoli cell number. Interstitial compartment volume was greater (P < 0.01) in hemicastrated boars. Leydig cell numbers were similar (P = 0.14) but volume was greater (P < 0.01) for hemicastrates. There were no differences (P > 0.05) between control and hemicastrated boars in TGFalpha or FGF2 expression on Day 5 or Day 10, and EGF was not detected. It was concluded that upregulation of TGFalpha or FGF2 expression is not a pre-requisite for enhanced testicular growth and increased Sertoli cell proliferation that occurs subsequent to hemicastration in the neonatal boar.

Methods for quantifying follicular numbers within the mouse ovary.

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 +/- 286 (S.E.M.) and 2227 +/- 101 primordial follicles, and 137 +/- 25 and 265 +/- 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.

Marked extension of proliferation of rat Sertoli cells in culture using recombinant human FSH.

Previous studies indicate that proliferation of rat Sertoli cells in culture can only be maintained until the equivalent of days 10-12 after birth, irrespective of the age of the donor animal. This report describes methods for the isolation and culture of Sertoli cells from day 6 rat testes, which can proliferate in culture for 20-24 days (that is, until the equivalent of days 26-30 after birth). Cells were isolated by enzymatic digestion of seminiferous cords followed by selective depletion of contaminating peritubular cells by adhesion to a polystyrene surface. The purity of the Sertoli cells was assessed using a combination of markers to be > 99.5%. Proliferation was assayed using tritiated thymidine incorporation and further verified by bromodeoxyuridine histochemistry and flow cytometry. Sertoli cells proliferated at basal levels in Dulbecco's modified Eagle's medium (DMEM)-F12 media alone, and proliferation was stimulated further by addition of recombinant human FSH to the culture media. After 20-24 days in culture, proliferation rapidly ceased, and cells assumed abnormal morphology and detached from the culture vessel; these events are consistent with the cells undergoing classic rodent cell senescence. The method described provides a useful tool for investigating the control of Sertoli cell division. Furthermore, these findings indicate that the timely differentiation of Sertoli cells is not dependent solely on an intrinsic timing mechanism, as has been suggested previously.

Resistance of the dentate gyrus to induced apoptosis during ageing is associated with increases in transforming growth factor-beta1 messenger RNA.

Up-regulation of endogenous neurotrophic factors may protect against apoptosis during ageing. Recent studies showed that the expression of several neurotrophic factors increased with age in specific regions of the rat brain. Previously, we showed that removal of trophic adrenal steroids by adrenalectomy induced apoptosis in the dentate gyrus of adult rats, which was accompanied by increased expression of transforming growth factor-beta1 (TGF-beta1) messenger RNA. In this study, we compared the relative densities of apoptotic cells in the dentate gyrus with TGF-beta1 messenger RNA expression in virgin male Fischer 344 rats ranging from 2 to 26 months of age across three treatment groups: adrenalectomy, adrenalectomy with corticosterone replacement, or sham operation. Seven days after adrenalectomy an increase in the density of apoptotic cells was observed in rats of all age groups compared with sham-operated and corticosterone-treated groups. By in situ hybridisation, the glial messenger RNAs, TGF-beta1 and glial fibrillary acidic protein as a marker of ageing, increased following adrenalectomy in the dentate gyrus in rats of all ages compared with control groups. Interestingly, within adrenalectomy groups, both the number and density of apoptotic cells decreased significantly by 6-8 months with a further decrease at 24-26 months of age. Furthermore, the amount of apoptosis corresponded to changes in TGF-beta1 expression, notably in: (i) adrenalectomised rats showing a significant inverse correlation and (ii) 24-26-month-old rats with the lowest induced apoptosis showing increased expression at the time of adrenalectomy. These studies show that resistance to adrenalectomy-induced apoptosis in the dentate gyrus is associated with increases in TGF-beta1 messenger RNA expression. Furthermore, the endogenous up-regulation of neurotrophic factors, such as the increase in TGF-beta1 expression in the oldest rats, suggests that the aged brain may have compensatory mechanisms, which protect against apoptosis.

Male reproductive phenotypes in double mutant mice lacking both FSHbeta and activin receptor IIA.

Activins are known to signal through two serine/threonine kinase type II receptors. Activin receptor IIA is widely expressed in the male reproductive axis, including the pituitary and testis. Our previous studies using gene knockout mice have confirmed the essential in vivo role of activin receptor IIA in FSH homeostasis. Activin receptor IIA-null male mice are fertile, have suppressed pituitary and serum FSH levels, and demonstrate a decrease in testis size as a result of reduced Sertoli cells and germ cells. Similarly, FSHbeta null male mice are fertile despite reduced testis size and Sertoli cell number. To define the direct roles of activin receptor IIA signaling locally in the testis, independent of its effects on FSH homeostasis, we generated double mutant mice lacking both activin receptor IIA and FSH by a genetic intercross and analyzed the male reproductive phenotypes. The double mutant male mice lacking both FSH and activin receptor IIA are fertile, demonstrate no significant reduction in testis size, and produce small litters compared with mice lacking either FSH or activin receptor IIA alone. Histological analyses of the testes from double mutant mice revealed the presence of normal stages of spermatogenesis. However, there was a significant reduction in the epididymal sperm number compared with that of the individual mutants. Northern blot analyses of total RNA from testes of double mutants did not reveal transcriptional up-regulation of activin receptor IIB, the other activin type II receptor. Although RNA expression profiles of many testis cell-specific markers are unaltered, stereological analysis of the testes from double mutants indicates that there was a reduction in type A and I spermatogonial number compared with that observed in individual mutants. Our results provide in vivo genetic evidence to demonstrate that activin receptor IIA signaling plays an important local role within the testis, independent of its actions via FSH homeostasis in the pituitary.

Inhibins, activins and follistatin: actions on the testis.

While the early studies of the inhibins, activins and follistatins concentrated on their role as endocrine regulators of FSH secretion, recent data has emphasized the local actions of the activins and follistatin. Inhibin, through its capacity to suppress FSH secretion can modulate numerous processes within the testis. However, to date, evidence to support a local role for inhibin is limited. In contrast, activin and its binding protein follistatin are produced by a large number of cell-types within the testis raising the possibility of a range of paracrine and autocrine actions. These include the modulation of androgen production, influence on the proliferation of Sertoli cells and germ cells as well as the capacity to influence the structural and functional features of mitochondria within germ cells. Some of these actions are carefully controlled in a temporal relationship during the development of testicular function in the rat in which there is no separation in time between birth and the onset of spermatogenesis. Given the range of actions of activin in different cell-types, recognition of systems that are designed to modulate its actions are crucial in enhancing our understanding of how these many roles can be compartmentalized.

Analysis of the testicular phenotype of the follicle-stimulating hormone beta-subunit knockout and the activin type II receptor knockout mice by stereological analysis.

This study evaluated the role of FSH and activin A on testicular function using quantitative stereological analysis of testicular cell types in mice with targeted disruption of genes encoding the FSH beta-subunit and the activin type IIA receptor (ActRIIA). Using the optical dissector technique, the numbers of Sertoli cells and germ cells per testis were determined. Testis weights in homozygous males lacking the FSHbeta gene or the ActRIIA gene were decreased approximately 60% compared with wild-type or respective heterozygotes. Sertoli cell numbers decreased in both homozygous mice by 30-39%, and there was a comparable decline in germ cell numbers in both models. The degree of germ cell attrition increased in the later stages of spermatogenesis from a 46% reduction of spermatogonia to a 60% decrease in round spermatids. As the FSH levels are decreased in both models, the cellular lesion in both is most likely due to the FSH deficiency. Although the decrease in the Sertoli cell complement represents one cause of lower germ cell numbers, the ability of Sertoli cells to nurture germ cells is compromised by the lower FSH levels, as shown by a decrease in the round spermatid to Sertoli cell ratios in both homozygous models. We conclude that the defects in FSH beta-subunit gene knockout and ActRIIA knockout mice are related to diminished FSH action on both Sertoli cell proliferation and the capacity of Sertoli cells to nurture germ cells.

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.

Aromatase-deficient (ArKO) mice accumulate excess adipose tissue.

Aromatase is the enzyme which catalyses the conversion of C19 steroids into C18 estrogens. We have generated a mouse model wherein the Cyp19 gene, which encodes aromatase, has been disrupted, and hence, the aromatase knockout (ArKO) mouse cannot synthesise endogenous estrogens. We examined the consequences of estrogen deficiency on accumulation of adipose depots in male and female ArKO mice, observing that these animals progressively accrue significantly more intra-abdominal adipose tissue than their wildtype (WT) litter mates, reflected in increased adipocyte volume and number. 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 to WT controls, as were elevated insulin levels, although blood glucose was unchanged. Associated with these changes, the livers of ArKO animals were characterised by a striking accumulation of lipid droplets. Our findings demonstrate an important role for estrogen in the maintenance of lipid homeostasis in both males and females.

The ovarian phenotype of the aromatase knockout (ArKO) mouse.

Targeted disruption of exon 9 of the cyp19 gene gives rise to a non-functional aromatase enzyme incapable of converting androgens to oestrogens. The aromatase knockout (ArKO) mouse is, thus, characterised by a dysfunctional pituitary-gonadal axis, which manifests in non-detectable levels of oestrogen in serum. These mice also exhibit elevated levels of circulating gonadotrophins (luteinising hormone (LH) and follicle stimulating hormone (FSH)) and testosterone. The ArKO mouse is infertile due to folliculogenic disruption and a failure to ovulate. The age-dependent ovarian phenotype revealed a block in follicular development at the antral stage and a complete absence of corpora lutea. By 21-23 weeks of age haemorrhagic cystic follicles were present and by 1 year there were abnormal follicles, an absence of secondary and antral follicles and atretic primary follicles. Interstitial tissue remodelling was extensive and exemplified by an increase in collagen deposition and an influx of macrophages, coincident with the loss of follicles. In mice, maintained on a soy-free and, thus, phytoestrogen-free diet, the ovarian phenotype was accelerated and exacerbated. In conclusion, the ovarian phenotype of the ArKO mouse can be attributed to the altered hormonal environment brought about by the absence of aromatase and the failure of androgens to be converted to oestrogens in the presence of elevated gonadotropins.

Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity.

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.

The roles of activins, inhibins and estrogen in early committed follicles.

The hypothesis that activin and inhibin are autocrine/paracrine mediators of ovarian folliculogenesis has a solid basis. In mouse and rat models, granulosa cells (GC) of committed follicles express mRNA and protein for the activin/inhibin subunits and mRNA for the activin receptors (type I and II). Dimeric inhibin-A and -B are produced by postnatal ovarian cell dispersates and (GC) in culture. Similar levels of inhibin-A and -B are produced by postnatal ovarian cells, but thereafter as the ovary develops, inhibin-A becomes the predominant form. Activin was more effective than transforming growth factor-beta (TGF-beta) in enhancing follicle stimulating hormone (FSH)-stimulated inhibin production by ovarian cells. Evidence for a local regulatory role of estrogen in the ovary is also accumulating. Murine models of estrogen receptor (ERalpha or ERbeta) disruption produce mice with abnormal ovarian phenotypes. Female mice, which lack the capacity to produce estrogen (ArKO mice), have arrested folliculogenesis, no corpora lutea, elevated levels of luteinising hormone (LH), FSH and testosterone and are infertile. These data are consistent with autocrine/paracrine actions of activin in the early growth of committed follicles and estrogen in follicular maturation.

An age-related ovarian phenotype in mice with targeted disruption of the Cyp 19 (aromatase) gene.

With the development of a mouse model of estrogen insufficiency due to targeted disruption of the aromatase gene [the aromatase knockout (ArKO) mouse], a new opportunity exists to examine the role of estrogen in ovarian follicular development. Ovaries and serum were collected from wild-type, heterozygous, and ArKO mice at 10-12 and 21-23 weeks and 1 yr of age. The ovaries were assessed histologically and stereologically, with primary, secondary, and antral follicles and corpora lutea counted. The uteri were hypoestrogenic, and serum levels of LH and FSH in ArKO females were elevated above those in heterozygote and wild-type animals at all ages studied. Although estrogen was not a prerequisite for reinitiation of follicle growth, there was a block of follicular development, and no corpora lutea were present in ArKO ovaries. Thus, the ArKO mouse was infertile as a consequence of disrupted folliculogenesis and a failure to ovulate. Hemorrhagic cystic follicles were present by 21-23 weeks of age. The ovarian phenotype degenerated with age, such that by 1 yr there were no secondary or antral follicles, and the primary follicles present were atretic. Extensive interstitial tissue remodeling occurred, exemplified by an influx of macrophages and collagen deposition, coincident with the loss of follicles. In conclusion, the ovarian environment in ArKO mice does not allow the characteristic development of follicles that culminates in ovulation and demonstrates an in vivo requirement of estrogen for normal ovarian function in the mouse.

Developmental expression of thyroid hormone receptors in the rat testis.

Sertoli cell proliferation in the rat is completed by Days 15-20 postnatally. Thyroid hormones appear to regulate the duration of Sertoli cell proliferation, affecting adult Sertoli cell number and hence the capacity of the testis to produce sperm. In the present study, a combination of immunohistochemistry, immunoblot analysis, and reverse transcription-polymerase chain reaction was used to demonstrate the expression pattern of thyroid hormone receptors (TR) in the juvenile and adult rat testis. The results indicated that TRalpha1 was expressed in proliferating Sertoli cell nuclei, its expression decreasing coincident with the cessation of proliferation. TRalpha2, TRalpha3, and TRbeta1 mRNAs were expressed at low levels during development; however, the corresponding protein was not detected by immunoblot analysis. In addition, TRalpha1 was found to be expressed in germ cells from intermediate spermatogonia to mid-cycle pachytene spermatocytes. Immunohistochemistry also demonstrated TR expression in a subset of interstitial cells. The demonstration of TR expression in germ cells undergoing spermatogenic differentiation suggests a possible role for thyroid hormones in the adult testis.

FSH immunoneutralization acutely impairs spermatogonial development in normal adult rats.

Follicle-stimulating hormone (FSH) plays an important part in testicular development. Its role in the regulation of spermatogenesis in the adult, however, remains controversial. This study aimed to explore the role of FSH in the maintenance of adult rat spermatogenesis by using immunoneutralization to selectively withdraw FSH action for periods of up to 8.5 days and then assessing the outcome by quantification of germ cell number. Adult rats received either an ovine polyclonal rat FSH antibody (FSHAb, 2 mg/kg subcutaneous daily-a dosage known to neutralize >90% of FSH in serum) for 2, 4, 7, or 8.5 days or a control sheep immunoglobulin (ConAb, 2 mg/kg) for 8.5 days. Testes were perfusion fixed, and germ cell numbers per testis were quantified using the optical disector (sic) stereological method. The percentage of seminiferous tubules displaying apoptotic cells was determined by the in situ end labeling method (TUNEL). FSHAb treatment for 4, 7, or 8.5 days significantly reduced the number of type A/intermediate spermatogonia (approximately 74% of control values) associated with stages I-IV. Similar reductions were seen in type B spermatogonial and preleptotene spermatocyte numbers after 8.5 days of FSHAb treatment (approximately 69% of control values; P < 0.05). Decreases (P < 0.05) in the numbers of pachytene spermatocytes in stages I-III and VIII, round spermatids in stages I-III, VII, and VIII (approximately 70% of control values), and step 19 elongated spermatids in stage VII (51% of control values) were achieved after 8.5 days of FSHAb treatment. Compared with control, FSHAb treatment increased the percentage of stage XIV-III tubules containing TUNEL-positive cells by about twofold after 7 days of FSHAb treatment (P < 0.05). This study supports a role for FSH in the maintenance of quantitatively normal adult rat spermatogenesis, specifically by regulating A3 and A4 spermatogonial subtypes. FSH may act on these spermatogonia by enhancing the stage-dependent survival of type A spermatogonia. Effects at other sites in spermatogenesis are suggested by the changes in spermatocyte and spermatid populations. However, to clarify these effects, selective FSH withdrawal would need to be prolonged until steady state had been achieved.

Changes in testicular steroidogenic acute regulatory (STAR) protein, steroidogenic enzymes and testicular morphology associated with increased testosterone secretion in bulls receiving the luteinizing hormone releasing hormone agonist deslorelin.

Testosterone secretion and the expression and relative contents of steroidogenic acute regulatory (StAR) protein and steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (P450SCC), 3beta-hydroxysteroid dehydrogenase/delta(5)-->delta(4)-isomerase (3 beta-HSD), and (17)alpha-hydroxylase cytochrome P450/C17-20 lyase (P450(17)alpha) were determined in testicular tissues of bulls treated with a LHRH agonist. Testis morphology and spermatogenesis were also examined. In Experiment 1, bulls (30-mo-old) received no treatment (control, n = 7) or were implanted for 10 days with the LHRH agonist deslorelin (n = 7). Bulls were castrated on Day 10 and testis tissues prepared for Western and Northern blotting. At castration, bulls implanted with deslorelin had greater plasma testosterone (5-fold) and testis content of testosterone (10-fold) compared with control bulls. Relative content (per micrograms total testis protein or RNA) of StAR protein, 3beta-HSD, P450SCC, and mRNA for P450(17)alpha in bulls treated with deslorelin ranged from 3- to 6-fold that of control bulls. In Experiment 2, bulls (20-mo-old) were left untreated (control, n = 6) or implanted with deslorelin (n = 12) for 120 days. On Day 120, bulls were castrated and right testis tissues prepared for morphology. Testis volume and weight were increased (P < 0.01) in bulls treated with deslorelin compared with control bulls. Stereological analysis revealed that this increase occurred in all compartments (seminiferous epithelium, lumen and interstitium) studied, but was significant (P < 0.01) only for the seminiferous epithelium. Absolute numbers of round spermatids per testis were increased (P < 0.05) in bulls treated with deslorelin compared with control bulls. Increased testosterone secretion in bulls treated with deslorelin was associated with increased testicular StAR protein and steroidogenic enzymes. Bulls treated long-term with deslorelin had a faster rate of testis growth and increased daily sperm production at the end of the experiment.

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.

Stereological evaluation of human spermatogenesis after suppression by testosterone treatment: heterogeneous pattern of spermatogenic impairment.

Testosterone (T) treatment suppresses gonadotropin levels in normal men and is a promising reversible contraceptive that induces azoospermia in approximately 70% of subjects and oligospermia in the remainder; however, the basis of this variable response is unclear. This study aimed to investigate this reported variable response by examining the spermatogenic process and quantitating germ cell number in men after T-induced gonadotropin withdrawal. Ten normal fertile men (31-46 yr), already planning to undergo vasectomy, either received T enanthate (200 mg, i.m., weekly) for 19-24 weeks (n = 5; TE group) or proceeded directly to surgery (n = 5; controls), at which time a unilateral testicular biopsy was taken, and germ cell numbers were estimated using the optical disector stereological method. In response to TE treatment, serum T levels rose 2-fold, and FSH/LH levels became undetectable. Sperm counts fell to azoospermia in 4 men and to 21 million/mL in the fifth man. The mean number of type A spermatogonia per 100 Sertoli cells was unchanged, but type B spermatogonia fell markedly to 10% of the control values, and later germ cell types decreased to 11-18% of the control values. The pattern of germ cell suppression varied widely and showed no relationship with sperm count or the time to azoospermia. Despite the presence of elongated spermatids (1.4-20% of the control), four men remained azoospermic. Two TE subjects with similar early germ cell complements and elongated spermatid numbers had sperm counts of zero and 21 million/mL; the latter man demonstrated marked variability in germ cell numbers between adjacent tubules. We conclude that 1) the principal spermatogenic lesion in TE-treated men is the marked (90%) inhibition of type A-->B spermatogonial maturation. Other sites are also affected, particularly the release and/or survival of elongated spermatids during transit; and 2) a steady state in germ cell number may not be established even after 4-5 months of TE treatment. The findings suggest that TE treatment does not adequately or consistently withdraw hormonal support for spermatogenesis, leading to variable between- and within-individual patterns of germ cell suppression.