Effect of local heating of rat testes after suppression of spermatogenesis by pretreatment with a GnRH agonist and an anti-androgen.

The effects of local heating of rat testes, in which spermatogenesis had been suppressed with injections of a GnRH agonist and an anti-androgen, were examined. Although the detrimental effects of heating were not as marked as those found in the testes of non-injected rats, the testes in which spermatogenesis was suppressed also showed a significant reduction in mass, the number of spermatozoa, tubular diameter and the percentage of normal tubular cross-sections at day 35 after heating. The results indicate that heating has an effect on cells in the testis other than those shown to be most susceptible to heat, namely pachytene spermatocytes and early spermatids, which were absent or markedly reduced in number when spermatogenesis was suppressed. The long-term effects of heating on the above parameters, as reported in a previous study, were also confirmed. However, in testes in which spermatogenesis was suppressed at the time of heating, there appeared to be no or a reduced long-term impairment of spermatogenesis, as determined by testis mass, the percentage of qualitatively normal tubules and epididymal sperm counts.

Percutaneous cutting needle biopsies for histopathological assessment and sperm retrieval in men with azoospermia.

BACKGROUND: Twenty-three men (45 testes) with azoospermia underwent percutaneous testicular biopsy under local anaesthesia. METHODS: In all but one of the 45 testes two biopsies were taken close to each other, one with a 16 gauge (n = 44) and another with a 14 gauge (n = 45) cutting needle, both with a 19 mm notch. Three quarters of the tissue was used for histopathological assessment and one quarter for direct microscopy. RESULTS: The histopathological findings were similar between the two needles. The observations with direct microscopy corresponded with the histopathological assessments concerning the presence of mature spermatids in 41 of 45 (91%) biopsies using the 14 gauge and in 40 of 44 (91%) biopsies using the 16 gauge needle. There were no post-operative complications except for minimal pain and minor local swelling. CONCLUSIONS: Percutaneous material retrieved using 16 gauge and 14 gauge needles is sufficient for histopathological assessment, and the two needles are equally reliable for testicular sperm retrieval. However, needle biopsy with one puncture may not be representative of the entire testis.

Reduction of long-term effects of local heating of the testis by treatment of rats with a GnRH agonist and an anti-androgen.

Heating the testes of anaesthetized adult rats to 43 degrees C for 30 min in a waterbath was followed by a large decrease in testis and epididymis mass and number of spermatozoa 35 days later. These parameters had recovered to some extent, but not completely, by days 70 and 97 after heating, but had decreased again in rats examined on day 182. There were no consistent effects of heating on androgen status, as determined by the concentrations of testosterone in blood and testis fluids, or by seminal vesicle mass, and interstitial fluid volume was increased in the heated testes. Treatment of rats with an implant of a GnRH agonist and daily injections of an anti-androgen for 14 days (sufficient in itself to cause large temporary decreases in tissue mass, number of spermatozoa and androgen status) did not reduce the initial decrease in testis mass or number of spermatozoa seen after heating, but reduced the later decreases in mass and number of spermatozoa significantly. These findings indicate that, as well as causing damage to spermatocytes and spermatids, as previously reported, heating also reduces the ability of spermatogonia to repopulate the seminiferous tubules at longer intervals after heating. Furthermore, it appears that this effect on the spermatogonia can be reduced by treating the animals with a GnRH agonist and anti-androgen, a treatment similar to that shown by other authors to improve recovery of the testis from irradiation or drug treatment.

Tissue distribution of bovine cystatin C analysed by in situ hybridisation.

Cysteine proteinase inhibitors of the cystatin superfamily have been identified in many living organisms. However, knowledge of the tissue distribution of such inhibitors is limited. To elucidate this distribution in mammals, we have investigated the expression of the gene for cystatin C, belonging to cystatin family II, in several bovine tissues. In situ hybridisation with a digoxigenin-labelled cRNA probe demonstrated a high concentration of bovine cystatin C mRNA in the secretory epithelial cells of the choroid plexus, and also intense staining in cells of lymphoid tissue and in Sertoli cells. Cystatin C mRNA was also present in scattered neurons and glial cells throughout the cerebrum and the cerebellum. In the submandibular gland, specific mRNA was found mainly in striated intralobular ducts and interlobular ducts. The expression of cystatin C in brain tissue is of particular interest, as the inhibitor appears to be involved in certain neurological diseases. The main production of cystatin C within the brain is believed to be by astrocytes. However, this work shows that also neurons from young, normal individuals express cystatin C.

Improvement of spermatogenesis in adult cryptorchid rat testis by intratesticular infusion of lactate.

In order to test the hypothesis that a lack of energy could be a cause of germ cell death at high temperatures, cryptorchid rats testes were infused with lactate, delivered by osmotic pumps over 3-15 days. In cryptorchid testes, the spermatids and spermatocytes were lost between 3 and 8 days. In cryptorchid testes supplemented with lactate, elongated spermatids persisted in a few seminiferous tubules at Day 15. Elimination of round spermatids occurred progressively between 3 and 15 days, mostly at stage VIII. The loss of spermatocytes increased after 8 days, and 30% of seminiferous tubules still contained meiotic or meiotic plus spermiogenetic cells at Day 15. After 8 days, the chromatin of step 8 round spermatids was abnormal and nuclear elongation did not commence. The Sertoli cell cytoplasm that was retracted toward the basal compartment of the seminiferous epithelium could not hold the germ cells of the adluminal compartment. Therefore, attachment of germ cells to Sertoli cells and the supply of lactate seem necessary for the development of germ cells at high temperatures. The improvement in spermatogenesis in cryptorchid supplemented testes for several days is a new finding.

A comparison between open and percutaneous needle biopsies in men with azoospermia.

Open testicular biopsy is a classic method of investigation in men with azoospermia. Recently, percutaneous needle biopsy of the testis has been used in attempts to obtain material for histopathological diagnosis in such cases and to retrieve spermatozoa for intracytoplasmic sperm injection (ICSI). To determine whether a 19 gauge (G) and a 21G butterfly needle could be used for percutaneous aspiration of testicular tissue to determine the presence of mature spermatids and assess spermatogenesis, 10 patients (16 testes) and 12 patients (17 testes) underwent 19G or 21G needle biopsy respectively, immediately followed by open testicular biopsy, with both procedures under local anaesthesia. Biopsy with each needle size was compared with open biopsy. With the 19G needle, in the 14 cases where material was obtained there was full agreement with open biopsy regarding the presence or absence of mature spermatozoa, whereas with the 21G needle only nine of the 13 biopsies yielding material were predictive in this respect. Each needle size correlated poorly with open biopsy regarding evaluation of spermatogenesis. We conclude that percutaneous biopsy with a 19G butterfly needle is a quick and reliable method for demonstrating spermatozoa for ICSI. But for a detailed histopathological diagnosis, however, the needle biopsies gave poor results, whereas the material from the open testicular biopsies was assessable.

Effects of very high doses of human growth hormone (hGH) on the male reproductive system in the dog.

The effects of very high doses of human growth hormone (hGH), pituitary derived or recombinant methionyl-hGH, on the morphology of reproductive organs and on some hormones in the male dog are described. The studies were part of a toxicological documentation of hGH. A total of 18 male dogs aged 7.5-20.5 months, from four studies were treated subcutaneously with hGH for 20-28 days at dose levels of 3, 10 or 25 IU kg-1 day-1 or 1 IU kg-1 three times weekly. Plasma levels of luteinizing hormone (LH), testosterone and prolactin were determined in one study. Organ weighing, macroscopic and histopathologic examinations of male reproductive organs at the end of the treatment period were included in all studies. Treatment with 25 IU kg-1 day-1 resulted in reduction of testis and prostate weights, degeneration of germ cells and epithelial atrophy in the testis, degenerative changes in epididymis and reduced height of the prostatic epithelium. Similar, although less severe morphological changes were observed after treatment with 10 IU kg-1 day-1. Treatment with 25 IU kg-1 day-1 also caused a marked reduction of plasma prolactin, LH and testosterone levels. These results suggest that repeated administration of very high doses of hGH interferes with the hormonal regulation of the testis in the dog.

Reduction in fluid secretion by rat testis by drugs that block potassium channels.

The effect of two class III antiarrhythmic drugs (Almokalant, Astra-Hässle and Dofetilide, Pfizer) on fluid secretion by rat testes has been examined. Both drugs reduced fluid secretion, whether this was measured by the amount of rete testis fluid that could be collected 22 h after unilateral efferent duct ligation, or by the difference in mass between the ligated and unligated testes, or by the difference in amount of supernatant fluid after the parenchyma of the ligated and unligated testes had been dispersed and centrifuged. The secretion of potassium, calculated from the amount of potassium in the supernatant fluids from the ligated and unligated testes was also reduced by the drugs, whereas the secretion of androgen-binding protein and inositol was unaffected. The concentration of potassium in the secreted fluid, calculated from the amount and composition of the supernatant fluids, was not affected by treatment of the rats with Almokalant, but was increased in rats treated with Dofetilide and, in these, the concentration of sodium was reduced and that of magnesium and inositol was increased and the concentration of total protein was unaffected. The concentration of androgen-binding protein in secreted fluid was increased in rats treated with Almokalant, while the concentration of testosterone was unaffected. Histological examination of testes from treated rats revealed phagocytosis of stage 19 spermatids in tubules at stages VIII-IX after 2 days, at stages IX-XI after 4 days and at stages VIII-XIV after 7 days, apparently owing to an effect on spermiation. It appears that these drugs interfere with potassium-mediated fluid secretion by the testis, leading to the other changes seen.

Embedding resin space, water contents and chromatin compaction in rabbit sperm nuclei: electron microscopic X-ray spectrophotometry of a brominated probe.

EPON labelled with bromide was used to embed ejaculated rabbit spermatozoa, with the hypothesis that it replaces cell water. X-Ray spectrophotometric microanalysis of sperm nuclei, of egg yolk (an internal standard containing roughly 50% water) and of surrounding embedding resin, revealed that a part of the bromide was bound to the biological components. These latter were saturated when bromide was added in higher concentrations, and the increase in measured bromide could be used to calculate absolute resin contents in sperm nuclei which gives a mean value of 22.62%. Most nuclei (60.80%) were well condensed and displayed a mean resin space close to 17% of the total nuclear volume. The less condensed nuclei had a mean resin space close to 28%. The use of an internal standard revealed that calculated values were underestimated by 4%.

Reciprocal chromosome translocation, rcp(7;17)(q26;q11), in a boar giving reduced litter size and increased rate of piglets dying in the early life.

Cytogenetic investigations in a boar causing a 41% reduction in litter size and also producing piglets that died soon after birth revealed the presence of a reciprocal chromosome translocation, rcp(7;17)(q26;q11). The translocation resulted in one extremely small telocentric chromosome marker. Synaptonemal complex analysis of spread spermatocytes by electron microscopy revealed an unusual behaviour of the translocation. This formed not only different types of quadrivalents (78.1% of the cells), similar to those previously found in boars heterozygous for reciprocal exchanges, but also completely or incompletely paired trivalent configurations, plus univalent (21.9%). Association between the sex bivalent and the translocation configuration was observed (18.7%), but testicular histology was normal. Furthermore, the boar with the translocation was found to produce tertiary monosomy and trisomy in some of the liveborn piglets. Some of the tertiary monosomic offspring, which died in the early extra-uterine life, demonstrated ventricular septal defect and cleft palate.

Synaptonemal complex analysis in a boar with tertiary, trisomy, product of a rcp(7;17)(q26;q11) translocation.

Synaptonemal complex analysis by electron microscopy showed a trivalent formed by pairing of chromosome 17(7) with chromosome pair 7 in two zygotene cells. In 89 pachytene cells (75.4%) the chromosome 17(7) occurred as a univalent with the axis being unpaired, in the shape of a ring (60.6%), a rod (38.2%) or self-paired (1 cell). Thirty-four (38.2%) of the univalents were paired or associated with the sex bivalent. Twenty-five (21.2%) of the 118 pachytene cells analysed demonstrated different types of trivalent pairing: the 17(7) chromosome paired with chromosome pair 7 in 24 cells and with pair 17 in one cell. Moreover, in 4 cells, chromosome 17(7) was paired with both pair 7 and pair 17, forming a pentavalent. Trivalent association with XY was observed in 4 cells. Nineteen bivalents plus a univalent (8 cells), and eighteen bivalents plus a trivalent (11 cells), were found during conventional meiotic investigation of diakinesis-metaphase I. Though the boar demonstrated a normal testicular histology, a qualitatively deviant semen picture was evident.

Ultrastructural immunolocalisation of histones (H2B, H3, H4), transition protein (TP1) and protamine in rabbit spermatids and spermatozoa nuclei. Relation to condensation of the chromatin.

The histones H2B, H3 and H4, the transition protein TP1 and protamine were localised using ultrastructural immunocytochemistry in nuclei of rabbit spermatids and spermatozoa. Histones are present in round spermatid nuclei and are lost during the elongation of nuclei. TP1 and protamine appear simultaneously in all nuclei during this period. TP1 is located at the periphery of chromatin cords, while protamine seems to be located at random in the same cords. TP1 is lost in most elongated spermatids during step 13 of spermiogenesis, and the protamine stays in all sperm nuclei. TP1 remains present in some old spermatids and ejaculated spermatozoa. In the rabbit, 3-6% of sperm nuclei decondense spontaneously. Most are characterised by a retention of TP1. Respective roles of TP1 and the protamine in spermatid nuclear condensation are discussed.

Does HCG treatment induce inflammation-like changes in undescended testes in boys?

Twenty-two cryptorchid boys previously unsuccessfully treated with human chorionic gonadotropin (hCG) were operated. Testicular biopsies were taken and a routine orchidopexy was performed in each case. As controls eight cryptorchid boys without prior hormonal treatment were operated in the same way. A mild inflammation-like reaction was found in the cryptorchid testes in the period immediately following the last hCG injection. However, in testes studied 6 to 12 months after the last hCG injection there were no apparent signs of hCG-induced tubular damage.

Meiotic chromosome asynapsis in a boar with a reciprocal translocation and acquired testicular degeneration.

During a period of performance testing, a boar used for artificial insemination purposes was found responsible for reduced litter size and slightly increased incidence of repeat breeding. The qualitative and quantitative semen characteristics, however, were within normal limits. Later, evaluation of the semen during a period of 3 months revealed a decreased sperm concentration, but sperm cell morphology and mobility were normal. At castration, the boar, which had suffered from scrotal inflammation, was found to have hypoplastic gonads, the right testis being smaller than the left one. Histologically there was spermatogenic arrest at the primary spermatocyte level in almost all tubules of the right testis. In the left testis, histology was more heterogeneous, with some tubules containing all developmental stages, while others had an almost complete spermatogenic arrest. Cytogenetically the boar was carrying a translocation, rcp(2;14)(p14;q23). Synaptonemal complex analysis revealed complete quadrivalent pairing in 21 out of 43 cells analysed. The remaining cells demonstrated quadrivalents with axes moderately or extensively unpaired. There was a distinct difference between cells from the right vs. the left testis. The latter cells showed more completely paired translocation configurations. Moreover, in 14 and 7 cells analysed from the right and left testis, respectively, the translocation configuration could not be identified. This was due to chromosome asynapsis and frequent occurrence of gaps in the chromosomes, including others than those of the translocation, plus an abundant cellular deposit which formed a dense cell background. Association or pairing of the quadrivalent with the sex bivalent was seen in one cell only. At diakinesis-MI, most cells had a ring-shaped quadrivalent. It is believed that the asynapsis of chromosomes during the prophase of meiosis was to a large extent due to the degeneration caused by the inflammation processes.

Synaptonemal complex analysis of a reciprocal translocation, rcp(20;24) (q17;q25), in a subfertile bull.

A reciprocal translocation was identified in a subfertile artificial insemination bull. Somatic chromosome investigation of G-banded metaphases demonstrated a 60,XY,rcp(20;24)(q17;q25) karyotype for the carrier. Synaptonemal complex analysis of the translocation by electron microscopy revealed an irregular pairing behavior of the chromosome axes involved, which resulted in a variety of configurations at pachytene. Not only was the expected quadrivalent configuration present, but also a trivalent plus univalent and two heteromorphic bivalents. Most common was an incompletely or completely paired quadrivalent configuration, which was non-homologously paired. XY association with the multivalent was seen only rarely. Histological analysis of testicular tissue showed meiotic arrest in some tubules. However, the semen picture was normal.

Comparison of nuclear hydration in boar spermatozoa before and after freeze-thawing: contrast analysis of cells embedded in bromide-labeled medium.

Epon labeled with bromide was used to embed ejaculated and freeze-thawed spermatozoa, with the hypothesis that it replaces most of cell water. Image analysis of relative contrasts between sperm nuclei and the surrounding medium revealed that when used in low concentrations, bromide is mostly adsorbed to the nuclear structures. For higher concentrations, the chromatin is saturated, and the increase in contrast can be used to calculate relative differences in the hydration of nuclei. Boar sperm nuclei are more hydrated after freeze-thawing than before.

Synaptonemal complex analysis of an autosomal trisomy in the horse.

Synaptonemal complex analysis by electron microscopy of a trisomy 28 in a male horse demonstrated a trivalent or a bivalent plus a univalent in primary spermatocytes. Two of the chromosomes making up the trivalent were, most often, completely paired with each other and only partially paired or associated with the third one. Half of the spermatocytes analysed demonstrated heterologous pairing or association between the free axis of the trivalent and the sex bivalent. The pairings remained, to a large extent, into diakinesis-metaphase I. In most pachytene cells one autosomal bivalent showed proximal asynapsis and paired often, heterologously, with the trivalent or the sex bivalent. The horse demonstrated azoospermy, which was due, at least in part, to degeneration at both the spermatocyte and spermatid levels.

The fertility potential of male cystic fibrosis patients.

Men suffering from cystic fibrosis (CF) are considered to be infertile because of azoospermia. In testicular biopsies from two patients with CF normal spermatogenesis was found despite the absence of sperm in the ejaculate. Fructose and prostaglandin were not detectable in the semen whereas the levels of acid phosphatase and zinc were within normal limits, indicating normal prostatic function and absence of seminal vesicles. These findings may improve the possibility for male patients with CF to father a child.

Histochemical localization of carbonic anhydrase in the testis and epididymis of the boar.

The testis and epididymis of sexually mature, fertile boars were studied for localization of carbonic anhydrase (CA) using a cobalt precipitation technique. In the testis, cytoplasmic CA was found in the Sertoli cells and in the capillaries surrounding the seminiferous tubules. The epididymal duct was divided into initial, middle and terminal segments, and regional differences in CA activity were observed. The cell membranes of the basal cells were stained in the initial and middle segments. Strong cytoplasmic CA staining was present only in the apical cells in the initial and middle segments. The basolateral cell membranes were stained in the principal cells of the terminal segment and the ductus deferens. As a rule the capillaries surrounding the epididymal duct were unstained. The enzyme, specifically localized in regions of the male genitalia acting as sperm reservoirs, might be related to the quiescence of the stored spermatozoa by influencing the acid-base status of the epididymal fluid.