Biomedical subjects

G F Weinbauer

Publications and source records attributed to G F Weinbauer.

At least 37 records · Page 2Linked to original sources

PubMed1999

The Old World monkey DAZ (Deleted in AZoospermia) gene yields insights into the evolution of the DAZ gene cluster on the human Y chromosome.

The DAZ gene cluster on the human Y chromosome is a candidate for the Azoospermia Factor (AZFc). According to the current evolutionary model, the DAZ cluster derived from the autosomal homolog DAZL1 through duplications and rearrangements and is confined to Old World monkeys, apes and humans. To study functional and evolutionary aspects of this gene family we have isolated from a cynomolgus (Old World) monkey testis cDNA library the Y chromosomal cynDAZ and the autosomal cynDAZL1 cDNA. cynDAZL1 contains one DAZ repeat and displays high homology to human DAZL1. cynDAZ comprises 11 repeats, each consisting of exons 7 and 8, whereas the human DAZ cDNA repeat units contain predominantly exon 7. Genomic studies revealed the same amplific- ation events of a 2.4 kb genomic unit encompassing exons 7 and 8 in both species, indicating that after splitting of the two lineages, in the human mainly exon 8 was converted to a pseudoexon by splice site mutations. The structural features of cynDAZ reveal a more detailed model for the sequence of events leading to the present form of human DAZ. Thus, in a monkey species DAZ is present in a form more ancestral than that of the human. Studies on the immunolocalization of cynDAZ / DAZL1 in cynomolgus monkey testis revealed a biphasic expression pattern with proteins being detectable in A-pale to B-spermatogonia, late spermatocytes and spermatids, but not in early spermatocytes and late spermatids. In contrast, in the marmoset monkey, an animal lacking DAZ, DAZL1 protein was only expressed in late spermatocytes and early spermatids. These findings point to an additional function of cynDAZ / cynDAZL1 during spermato- genesis in the Old World monkey not needed in the New World monkey.

Animals↗

PubMed1999

Germ cell transfer into rat, bovine, monkey and human testes.

Germ cell transplantation is a potentially valuable technique offering oncological patients gonadal protection by reinitiating spermatogenesis from stem cells which were reinfused into the seminiferous tubules. In order to achieve an intratubular germ cell transfer, intratubular microinjection, efferent duct injections and rete testis injections were applied on dissected testes of four different species: rat, bull, monkey and man. Ultrasound-guided intratesticular rete testis injection was the best and least invasive injection technique with maximal infusion efficiency for larger testes. Deep infiltration of seminiferous tubules was only achieved in immature or partially regressed testes. This technique was applied in vivo on two cynomolgus monkeys. In the first monkey a deep infusion of injected cells and dye into the lumen of the seminiferous tubules was achieved. In the second, transplanted germ cells were present in the seminiferous epithelium 4 weeks after the transfer. These cells were morphologically identified as B-spermatogonia and located at the base of the seminiferous epithelium. In summary, this paper describes a promising approach for germ cell infusion into large testes. The application of this technique is the first successful attempt of a germ cell transfer in a primate.

Animals↗

PubMed1999

Germ cell-specific cyclic adenosine 3',5'-monophosphate response element modulator expression in rodent and primate testis is maintained despite gonadotropin deficiency.

cAMP response element modulator (CREM) is an important component of the cAMP-mediated signaling pathway and is essential for differentiation of haploid male germ cells. In the rodent, testicular expression of CREM is believed to be controlled by FSH. We studied the expression pattern of CREM and gonadotropic control in the nonhuman primate and rodent testis. Adult cynomolgus monkeys (Macaca fascicularis) received daily either vehicle or the potent GnRH antagonist (ANT) cetrorelix for periods of 25 and 56 days. Rats were also exposed to vehicle or ANT for periods of 14 and 42 days. ANT treatment suppressed pituitary gonadotropin secretion, reduced testis size, and altered spermatogenesis. A rabbit polyclonal antibody raised against recombinant CREM tau and reacting with CREM alpha, -beta, -gamma, -tau1, and -tau2 at similar affinities was used for immunocytochemistry and Western blotting. CREM expression was seen in round spermatids, with highest levels during spermatogenic stages V-VII, but declined with progression of spermatid development in the primate. Similar observations were made for the rat testis. Thus, CREM expression was maximal at the onset of acrosome formation and was low or undetectable upon initiation of spermatid elongation in both species. A weak, but specific, CREM signal was seen in mid- to late pachytene spermatocytes and during meiotic division in both species. After ANT exposure, the germ cell- and stage-specific pattern of CREM expression was quantitatively retained at all time points and in both species. Northern and Western blot analysis confirmed the maintenance of testicular CREM expression despite 25 days of ANT treatment. A retrospective immunocytochemical analysis of rat testes 14 days posthypophysectomy revealed CREM signals in round spermatids. These findings demonstrate that the testicular expression of CREM is not entirely dependent on gonadotropic hormones but, rather, on the maturational stage of haploid round germ cells.

Animals↗

PubMed1999

Serum inhibin B in combination with serum follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesis in men, but cannot predict the presence of sperm in testicular tissue samples.

The measurement of serum FSH is useful in the diagnostic workup of the infertile male, but fails to predict the presence of sperm in testicular tissue. We investigated whether inhibin B reflects testicular morphology and the presence of sperm more accurately than FSH. Serum inhibin B and gonadotropin levels were determined in 91 infertile men undergoing diagnostic bilateral testicular biopsy. In 52 of the 91 patients multiple samples were taken for testicular sperm extraction (TESE). Inhibin B levels were (mean +/- SEM) 238+/-32 pg/mL in men with normal spermatogenesis (n = 9), 102+/-18 pg/mL in men with spermatogenetic arrest (n = 15), 98+/-16 pg/mL in hypospermatogenesis (n = 23), 41+/-6 pg/mL in focal Sertoli cell-only syndrome (SCO; n = 26), and 27+/-8 pg/mL in complete SCO (n = 18). The percentage of SCO tubuli was more strongly correlated to serum inhibin B (r = -0.58; P<0.01) than to FSH (r = 0.34; P<0.05). Similarly, the percentage of tubules with elongated spermatids was significantly (P<0.05) more strongly correlated to serum inhibin B (r = 0.65; P<0.01) than to FSH (r = -0.4; P<0.01). Thus, inhibin B is slightly more sensitive than FSH as an index of the spermatogenic status. Neither FSH nor inhibin B alone, however, could predict the type of spermatogenetic damage exactly. The combination of FSH and inhibin B had high diagnostic sensitivity (88%) and specificity (83%) for the presence of elongated spermatids in testicular biopsies. Sperm could be retrieved in 34 (65%) of the TESE patients. The combination of inhibin B and FSH measurement showed a sensitivity of 75% and a specificity of 73% when identifying patients in whom sperm could possibly be retrieved by TESE. We conclude that although the measurement of serum inhibin B improves the sensitivity of predictive tests for the presence of sperm in histology or for TESE, this parameter cannot accurately predict TESE outcome.

Adolescent↗

PubMed1999

Lack of effects of recombinant human GH on spermatogenesis in the adult cynomolgus monkey (Macaca fascicularis).

OBJECTIVE: The effects on male reproductive parameters after 1 year of treatment with recombinant human GH to the cynomolgus monkey were investigated. DESIGN: Twenty-four male cynomolgus monkeys were given daily subcutaneous doses of 0 (vehicle) (n=7), 0.4 (n=5), 2.0 (n=5) and 10.0 (n=7) IU/kg bodyweight for 52 weeks. At completion of the treatment period two control and two high-dose animals were left for a 12-week treatment-free period. METHODS: Before and during the treatment period and during the recovery period, sperm analyses, testicular volume measurements and hormone analyses of prolactin (PRL), LH, FSH, testosterone and IGF-I in serum, and analysis of serum antibodies against human GH were performed. Testicular morphology was monitored by biopsies, predose and on day 15 of the study, and with light microscopy on organ samples collected at time of death, at the end of the treatment, and during recovery periods respectively. RESULTS: Of all studied parameters, alterations were observed only in serum levels of IGF-I and PRL. IGF-I showed a dose-dependent increase throughout the treatment, with a normalisation during the treatment-free period. PRL decreased significantly in animals given 10.0IU/kg per day from week 14 of treatment and throughout the study but with a normalisation upon cessation of treatment. Spermatogenesis, as judged from semen analysis, testicular volume measurements and testicular morphology was not affected. CONCLUSION: This controlled preclinical study demonstrates that high doses of human GH do not alter male reproductive parameters in a non-human primate model.

Animals↗

PubMed1999

The cycle duration of the seminiferous epithelium remains unaltered during GnRH antagonist-induced testicular involution in rats and monkeys.

Although the gonadotropic control of the spermatogenic process is well established, the endocrine regulation of the timing and kinetics of germ cell development has received little attention. We found previously that the administration of a GnRH antagonist (ANT) over a period of 25 days could retard spermatid development and slightly prolong cycle length in intact adult cynomolgus monkeys (Macaca fascicularis). The aim of the present study was to investigate the effects of extended exposure to ANT on the duration of the cycle of the seminiferous epithelium in the monkey. Additionally, the duration of spermatogenesis was studied in the ANT-exposed rat model. In experiment 1, monkeys were given either saline or ANT (n=6/group) and on day 30 all animals received a single injection of 5-bromodeoxyuridine (BrdU) to label S-phase germ cells. Testicular biopsies were taken on days 39, 43, 47 and 51 (end of treatment) for BrdU localization and flow cytometric analysis. ANT treatment suppressed hormone levels, reduced testis size by >70% and severely impaired germ cell production. Despite these alterations, cycle duration remained unchanged at all time-points compared with controls (10.12+/-0.15 days vs 10.16+/- 0.44 days). In experiment 2, adult male Sprague-Dawley rats (n=15/group) received either vehicle (VEH) or ANT for 14 days and received BrdU injection on day 2. Cycle duration was found to be shorter in the ANT-treated group (12.45+/-0.09 days) than in the control group (12.75+/-0.08, P<0.05). As spermatogenic cycle length in this control group was longer than that of our historical controls (range: 12.37-12.53 days), experiment 2 was repeated (n=10/group). In experiment 3, cycle duration was 12.51+/-0.02 for VEH and 12.46+/-0.05 for the ANT-treated group (P>0.05) in both species. We concluded that the duration of the cycle of the seminiferous epithelium in monkeys and rats is independent of gonadotropins but is rather regulated by the spermatogenic tissue itself.

Animals↗

PubMed1999

Inhibin B is a more sensitive marker of spermatogenetic damage than FSH in the irradiated non-human primate model.

This study evaluated the effect of bilateral testicular irradiation (2 Gy) on reproductive hormones, testicular volume (TV) and sperm parameters in six adult cynomolgus monkeys. Hormone levels (FSH, inhibin B and testosterone (T)) were determined to find the most valuable endocrine marker of irradiation-induced damage. All parameters were analysed at weekly intervals for 14 weeks. Histological evaluation of both testes was performed at week 14 after irradiation when one monkey was castrated and at week 27 when the remaining five monkeys were bilaterally biopsied. A decrease in body weight, TV (30% of the pre-treatment size) and sperm count was observed after irradiation. Severe oligozoospermia was achieved throughout the study but azoospermia was recorded only occasionally. Histological evaluation revealed a heterogeneous picture with patchy arrangement of seminiferous tubules containing advanced germ cell types. An increase (P<0.05) in FSH levels and, to a lesser degree also in T levels, occurred several weeks after irradiation. Inhibin B levels showed a sharp decline (P<0.001) as soon as 1 week after irradiation. FSH and inhibin B did not return to baseline levels during the observation period. A negative correlation was found between FSH and inhibin B values (r=-0.35, P<0.001). Inhibin B correlated positively with testis volume (r=0.73, P<0.001) and sperm counts (r=0.55, P<0.01). In conclusion, this study shows that inhibin B represents an early and more sensitive marker of testicular damage than FSH. Furthermore, the rapid fall of inhibin B after irradiation suggests that this hormone is a direct parameter of premeiotic germ cell proliferation.

Animals↗

PubMed1999

Role of FSH in male gonadal function.

The production of male gametes depends on the concerted action of the two gonadotropins FSH and LH on the testis. The action of LH is mediated through the production of testosterone by the Leydig cells. Since male germ cells possess neither FSH nor androgen receptors, the action of FSH and testosterone occurs through the Sertoli cells. Although the precise function of these two hormones remains elusive, the existing evidence suggest that both FSH and testosterone are able to stimulate all phases of spermatogenesis. In the male FSH is required for the determination of Sertoli cell number, and for induction and maintenance of normal sperm production. The crucial role of FSH in male gonadal function has been clearly illustrated by the discovery of a patient with an activating mutation of the FSH receptor. This patient had been hypophysectomized because of a pituitary tumor and, under testosterone substitution was unexpectedly fertile in spite of undetectable serum gonadotropin levels and had fathered three children. In this patient we could demonstrate a heterozygous activating mutation of the FSH receptor which resulted in cAMP production independent of FSH stimulation. This finding represents the first description of an activating mutation of the FSH receptor and demonstrates that FSH alone maintains spermatogenesis in man. On the other hand, the effects of the lack of FSH action are unclear. Among five men with a homozygous inactivating mutation of the FSH receptor only one was infertile and spermatogenesis was variably affected in the others. However, serum inhibin B values in these men were not completely suppressed and serum FSH levels were only moderately elevated, indicating the possibility that FSH receptor function was not completely abolished by the mutation. Elimination of FSH action is a prerequisite to suppress completely spermatogenesis for contraceptive purposes, while administration of both LH and FSH is necessary to induce sperm production in patients with hypogonadotropic hypogonadism. Experimental immunization of male monkeys against FSH markedly reduced germ cell proliferation and even induced infertility. At the cellular level, FSH stimulates the cAMP-dependent activation of protein kinase A in Sertoli cells, but the molecular mechanism of FSH action is poorly understood. In the primate, the gonadotropin withdrawal achieved by administration of a GnRH antagonist leads to a premeiotic arrest of germ cell proliferation, probably due to inhibition of the mitotic division of A-pale spermatogonia. Therefore, FSH might be the prime inducer of spermatogonial proliferation, while the successive maturation process could proceed independently of FSH. In summary, clinical and experimental evidence support the concept of an irreplaceble role of FSH in the primate. Only the combination of FSH and testosterone, however, supports a qualitatively and quantitatively fully normal spermatogenesis.

Animals↗

PubMed1998

Hormonal regulation and germ cell-specific expression of heat shock protein 60 (hsp60) in the testis of macaque monkeys (Macaca mulatta and M. fascicularis).

Decrease of heat shock protein 60 (hsp60), a mitochondrial chaperonin, in germ cells of men has been shown to be associated with low spermatogenic efficiency. In the present study, we have investigated the hormonal regulation of hsp60 in a pre-clinical primate animal model. Hsp60 production in the testes of the intact cynomolgus monkey (Macaca fascicularis) and animals that had been treated with the GnRH antagonist Cetrorelix for 25 days was studied by immunohistochemistry. In addition, testes of untreated adult rhesus monkeys (Macaca mulatta) and immature animals either exposed to human chorionic gonadotrophin (hCG), human follicle stimulating hormone (FSH) or hCG and FSH in combination, as well as vehicle-treated controls were analysed. In adult monkeys, specific hsp60 staining was observed in Leydig cells, spermatogonia and early primary spermatocytes. The labelling in Sertoli cells was not stage dependent. The hsp60 staining pattern was unaffected by gonadotrophin releasing hormone (GnRH) antagonist treatment. Western blot analysis confirmed the presence of a single band of 60 kDa in testicular homogenates of the cynomolgus monkey. In the testis of immature rhesus monkeys, hsp60 immunoreactivity was visible in gonocytes, spermatogonia and in Sertoli cells, whereas interstitial cells were negative. In the experimental study, hCG alone or in combination with FSH caused a substantial and marked upregulation of the chaperonin in Leydig cells. Human FSH alone did not affect hsp60 expression. We conclude that hCG is an important regulator of Leydig cell hsp60 expression during development, whereas FSH in immature animals and GnRH in adult monkeys is of less importance.

Animals↗

PubMed1998

Testicular cAMP responsive element modulator (CREM) protein is expressed in round spermatids but is absent or reduced in men with round spermatid maturation arrest.

Mice lacking the functional cAMP responsive element modulator (CREM) gene, a component of cAMP-mediated signal transduction, exhibit a specific arrest of round spermatid development although follicle stimulating hormone (FSH) and androgen secretion are not impaired. We studied testicular expression of CREM protein by immunocytochemistry in four patients with complete spermatogenesis (obstructive azoospermia), in 20 infertile patients with round spermatid maturation arrest (n = 10) or mixed atrophy (n = 10) and in six prostate cancer patients undergoing orchidectomy. Concentrations of testosterone were below normal in three patients. Concentrations of luteinizing hormone (LH) were lowered in two patients and elevated in one patient. FSH concentrations were above normal in ten patients. During normal spermatogenesis, CREM was expressed in nuclei of round spermatids in stages I-III of spermatogenesis but not in elongating spermatids. Western blot analysis of testes from prostate cancer patients indicated a major CREM band of approximately 35 kDa. Among patients with predominant round spermatid maturation arrest, CREM expression was significantly reduced (P < 0.05) or undetectable as revealed by quantitative image analysis. CREM-negative spermatids failed to progress beyond stage III of spermatogenesis. Our observations suggest a role for CREM in human spermatid development and raise the possibility that altered CREM expression could be associated with spermatid maturation defects in some cases of idiopathic male infertility.

Adolescent↗

PubMed1998

Acute and specific impairment of spermatogonial development by GnRH antagonist-induced gonadotrophin withdrawal in the adult macaque (Macaca fascicularis).

This study examined the effect of GnRH-antagonist (GnRH-A)-induced gonadotrophin withdrawal on numbers of germ cells in adult cynomolgus monkeys and aimed to identify the site of the earliest spermatogenic lesion(s) produced. Animals received either GnRH-A (Cetrorelix; 450 micrograms kg-1 day-1 s.c.; n = 5) or vehicle (control, n = 4) for 25 days. One testis was removed on day 16 and the other testis on day 25. The optical disector stereological method was used to estimate germ and Sertoli cell numbers per testis. After GnRH-A treatment for 16 days, the number of type A spermatogonia was unchanged; however, type B spermatogonia (15% of control), preleptotene + leptotene + zygotene (15% control) and pachytene (55% control) spermatocytes were all reduced (P < 0.05). By day 25, these cells were further reduced together with step 1-6 spermatids (38% control; P < 0.05). More mature germ cells were unaffected. The proportion of type A pale spermatogonia at stages VII-XII was reduced (P < 0.05) in GnRH-A-treated groups (52% on day 16, 43% on day 25) compared with control (67%). After 25 days of GnRH-A treatment, the number of Sertoli cells was unaltered but nuclear volume was reduced (66% control, P < 0.05). Tubule length was unchanged but volume (50% control), diameter (62% control) and epithelial thickness (59% control) were reduced (P < 0.05). GnRH-A treatment suppressed serum testosterone concentrations into the castrate range, and testicular testosterone concentrations to 21-36% of control values. Serum inhibin (as an index of FSH action) was suppressed in GnRH-A-treated animals by day 16, declining to 38% of control concentrations at day 25. Therefore, the primary lesion produced by GnRH-A induced gonadotrophin withdrawal is the rapid and profound reduction in the number of type B spermatogonia. The time course of germ cell loss suggests the inhibition of type A pale spermatogonial mitosis as the primary mechanism. Later germ cell maturation, specifically meiosis and spermiogenesis, appears to proceed unaffected. The decline in late spermatocytes and spermatids by 25 days of GnRH-A treatment is attributed to a 'depletional wave' from the spermatogonial lesion. The fact that such marked spermatogenic disruption occurs in the face of substantial testicular testosterone concentrations implies a significant role for FSH in spermatogonial development.

Analysis of Variance↗

PubMed1998

Gonadotrophin-releasing hormone antagonist arrests premeiotic germ cell proliferation but does not inhibit meiosis in the male monkey: a quantitative analysis using 5-bromodeoxyuridine and dual parameter flow cytometry.

Meiosis constitutes a crucial phase of spermatogenesis since the recombination of genetic information and production of haploid round spermatids need to be achieved. Although it is well established that gonadotrophic hormones are required for completion of the spermatogenic process, little is known about the dynamic and kinetic aspects of development of spermatocytes into spermatids and its endocrine control in the primate. In this study, S-phase germ cells were labelled using 5-bromodeoxyuridine (BrdU) incorporation and were then followed throughout meiosis under normal conditions and following GnRH antagonist (ANT)-induced gonadotrophin withdrawal in a nonhuman primate model, the cynomolgus monkey (Macaca fascicularis). Adult animals received either vehicle (VEH, n = 4) or the ANT cetrorelix (n = 5) throughout 25 days. On day 7 all animals received a bolus injection of BrdU. A biopsy was performed after 3 h, one testis was removed 9 days later (day 16 of treatment) and the other testis after 18 days (day 25 of treatment). Serum testosterone and inhibin levels, and testis weight were reduced (P < 0.05) by ANT treatment. BrdU localized to pachytene spermatocytes 9 days after BrdU and to round spermatids 18 days after BrdU in both groups, demonstrating that BrdU-labelled pachytene spermatocytes had undergone meiosis. Flow cytometric analysis revealed that the relative number and number per testis of BrdU-tagged 2C and 4C cells were reduced significantly (P < 0.05) within 16 days of ANT treatment. Numbers of 1C cells were lowered by day 25. The cell ratio for 1C:4C was similar with VEH and ANT (P > 0.05). These findings indicate that ANT reduced the number of cells available for meiosis but did not alter the rate of transition into round spermatids. Unexpectedly, however, the stage-dependent progression of BrdU-tagged round spermatids was significantly (P < 0.05) retarded under ANT as seen from the frequency of tubules containing BrdU-labelled round spermatids. The average duration of spermatogenic cycle was slightly prolonged (9.8 days in the VEH group and 10.8 days in the ANT group (P = 0.09)). Since no atypical germ cell associations could be found, it remains unclear whether this slight prolongation is entirely due to altered spermatid progression or whether earlier phases are affected. We conclude for the nonhuman primate that (1) BrdU-labelling of premeiotic germ cells is suitable for tracing their meiotic transition into postmeiotic cells, (2) unlike in the rat, gonadotrophin suppression initially affects premeiotic cell proliferation and thus the number of cells available for meiosis, (3) the meiotic process continues quantitatively despite gonadotrophin deficiency and (4) prolonged gonadotrophin deficiency might alter the timing of germ cell development.

Analysis of Variance↗

PubMed1998

Regulation of gonadotrophin secretion by inhibin, testosterone and gonadotrophin-releasing hormone in pituitary cell cultures of male monkeys.

The effects of bovine inhibin, testosterone and GnRH on gonadotrophin secretion by primate pituitary cells were characterized in vitro using pituitaries from six male rhesus monkeys and one male cynomolgus monkey. The effect of inhibin on basal secretion of FSH and LH was investigated. Dose-response curves in monkeys and rats were compared. GnRH dose-response curves in the presence and absence of testosterone were also examined in monkeys. In monkey pituitary cells, testosterone at a concentration of 10(-7) M had no effect on LH or FSH secretion. Inhibin suppressed FSH secretion to 50.8% of that of controls with no effect on LH. In rats, FSH secretion was suppressed to 45.0% of that of controls with a median effective dose (ED50, 95% range) of 1.298 (1.064-1.584) U/ml, compared with 1.024 (0.7204-1.455) U/ml in monkeys. In monkey pituitary cells, LH release was stimulated 9.9-fold and FSH 3.3-fold by GnRH. Testosterone had no effect on basal or GnRH-stimulated gonadotrophin release. These results support the view that the pituitary is not the target organ for the negative feedback action of testosterone in the male. In vitro, inhibin is the major regulator of FSH secretion at the pituitary level.

Analysis of Variance↗

PubMed1997

Interaction of the human epididymal protein CD52 (HE5) with epididymal spermatozoa from men and cynomolgus monkeys.

A monoclonal antibody (CAMPATH-1 G) against the human lymphocyte surface protein CD52, which is similar to the epididymal secretion HE5, was used to ascertain the presence of this protein on maturing primate spermatozoa by flow cytometry. The percentage of human viable spermatozoa stained specifically with this antibody increased from sperm in spermatocoeles (0.5%), to the efferent ducts (3.8%), corpus (47.2%), and cauda (85.7%) epididymidis. Positive cells revealed staining mainly over the whole tail and postacrosomal region of the sperm head. Spermatozoa (approximately 10%) from both the efferent ducts and corpus epididymidis took up additional antigen when incubated with human distal cauda epididymidal plasma as a source of CD52, and 12-22% of human testicular sperm (from spermatocoeles) took up CD52 from human seminal plasma. In the cynomolgus monkey, nonspecific binding of control IgG was greater than that in human males and net CD52 staining was measurable only on approximately 30% of corpus sperm where it was mainly on the principal piece. Neither caput nor cauda sperm took up human CD52 upon incubation with human seminal plasma, but an additional 27% of corpus sperm expressed CD52. Such uptake of CD52 was drastically reduced, or did not occur, when seminal plasma had been fractionated by filtration through 0.1 microns filters (filtrate II) or 300,000 Da cutoff filters (filtrate III), respectively. Western blots revealed that CD52 contents were much reduced in filtrate II and nondetectable in filtrate III of seminal plasma. Similar reduction of CD52 in the filtrate of cauda epididymidal plasma indicates the association of this epididymal secretion with large molecular factors and suggests their involvement as carriers in the in vivo transfer of the secretion onto the epididymal sperm surface. The in vitro uptake of CD52 by some but not all immature sperm and the detection by Western blotting of much less CD52 in the corpus than the cauda luminal plasma suggest that the acquisition of this epididymal secretion by spermatozoa depends on their maturation status as well as the availability of the protein in the epididymal lumen.

Animals↗

PubMed1997

Endocrine control of germ cell proliferation in the primate testis. What do we really know?

The present chapter reviews current knowledge concerning hormonal regulation of gametogenesis in the primate testis. LH/testosterone and FSH are the prime regulators of primate spermatogenesis. Although either hormone is capable of stimulating all phases of the spermatogenic process including the formation of sperm, the combination of both hormones is necessary in most instances to achieve quantitatively normal germ cell numbers. Sertoli cell proliferation can also be induced by either hormone in juvenile monkeys. Evidence for differential effects of testosterone and FSH on gametogenesis, however, is lacking and a synergistic effect is observed when they are combined. Receptors for androgens and FSH occur exclusively on testicular somatic cells and, hence, the trophic effects of these hormones on germ cell numbers are indirect ones. Interestingly, both hormones seem to have a common target, the spermatogonial population but it is unknown how such an indirect albeit highly specific effect is mediated. Whether the trophic hormone action influences germ cell numbers via increased proliferation or decreased cell death or both remains to be seen. There is evidence to suggest that the local androgen requirements for primate spermatogenesis might be comparatively high.

Androgens↗

PubMed1997

Estimation of the duration of the cycle of the seminiferous epithelium in the non-human primate Macaca mulatta using the 5-bromodeoxyuridine technique.

A comparatively low yield of germ cells has been reported for the spermatogenic process in primates. Kinetic studies of spermatogenesis and the spermatogenic cycle are needed to investigate this phenomenon but require the application of radioactively labeled compounds or irradiation. We have therefore investigated the suitability of a non-radioactive approach, viz., administration of 5-bromodeoxyuridine, for the determination of the kinetics of the spermatogenic cycle in a non-human primate, the rhesus monkey (Macaca mulatta). Four adult in-season animals received a bolus of 33 mg/kg 5-bromodeoxyuridine, one testis from each monkey was removed 3 h later and the other testis after 10 days and 11 h. Tissue was fixed in Bouin's solution and embedded in Paraplast. 5-Bromodeoxyuridine was localized by immunogold-silver staining with a monoclonal antibody. PAS-hematoxylin counterstaining was used for spermatogenic stage identification. At 3 h, the leptotene and zygotene spermatocytes in stages VII-IX were the most advanced 5-bromodeoxyuridine-positive cells. At 10 days 11 h, the label had advanced and pachytene spermatocytes in stages VI-IX contained 5-bromodeoxyuridine. The duration of the spermatogenic cycle was 10.42+/-0.07 days (range: 10.25-10.62 days). Peritubular cells and interstitial cells were rarely 5-bromodeoxyuridine-positive, and Sertoli cells were consistently negative for 5-bromodeoxyuridine. Importantly, our kinetic data closely resemble those obtained by means of the application of irradiation for this macaque species. We conclude that administration of 5-bromodeoxyuridine represents a non-radioactive reliable approach for studying kinetic aspects of the spermatogenic process in primates.

Animals↗

PubMed1997

Apoptosis as a mechanism of germ cell loss in elderly men.

In comparison with other mammals, human spermatogenesis is known to be an inefficient process, of which germ cell degeneration is a normal part. This study was performed to determine the mechanism of cell death in the testes of elderly men. Testes from 20 patients undergoing orchidectomy for prostate cancer were fixed, sectioned and processed for the detection of apoptosis using the in situ end-labelling technique. In addition, the formation of DNA ladders, a hallmark of apoptosis, was also investigated. Occurrence of apoptosis was not confined to a particular germ cell population but comprised all types of germ cells. Sertoli cell apoptosis was not encountered. The numbers of degenerating germ cells were determined per standard reference area, but no significant relationship was found between the mean values and age or testis weight. Analysis of the median values for germ cell death per reference area suggested that apoptosis occurs in clusters within the testis but is a rare occurrence outside these areas. It is concluded that spontaneous apoptosis can mediate germ cell death in a variety of cell types in the aged human testis.

Aged↗

PubMed1997

cynDAZLA: a cynomolgus monkey homologue of the human autosomal DAZ gene.

A gene on the human Y chromosome, specifically deleted in azoospermic patients (DAZ: deleted in azoospermia), and a DAZ homologue (DAZH) on human chromosome 3, have been recently described. In the present work we report the isolation and characterization of the corresponding DAZH gene of the cynomolgus monkey (Macaca fascicularis), which we have named cynDAZLA (cynomolgus DAZ-like autosomal). Reverse transcription-polymerase chain reaction was used to amplify the monkey DAZ homologue, and sequence analysis revealed an open reading frame of 888 bp encoding 295 amino acids. Northern blot hybridization of different tissues to a probe derived from the cynDAZLA cDNA detected a transcript of 3.5 kb that, in the male, was expressed only in the testis. Comparison of the cynDAZLA sequence to autosomal DAZ homologues from human, mouse and Drosophila showed two RNA-recognition motifs (RRM) and the presence of only one DAZ consensus repeat compared with the seven repeats found in the human DAZ gene on the Y chromosome. The homology of the cynDAZLA cDNA compared with the human DAZH and mouse dazla cDNAs is 97.97 and 87.46% respectively. The identification of the monkey cynDAZLA enables further studies regarding the putative functions of DAZH, such as onset of expression and hormonal dependence of this gene.

Amino Acid Sequence↗