Correlation between spermatozoon numbers in spermiogram and seminiferous epithelium histology in testicular biopsies from subfertile men.

A new method for correlating testicular biopsies and spermiograms is proposed. The number of spermatogonia, round spermatids, and elongated spermatids per cross-sectioned tubule were calculated in the testes from 33 subfertile men and in 10 control normal testes. According to these quantitations, the testes in subfertile men were classified as testes with maturation arrest of spermatogenesis, testes with hypospermatogenesis, and testes with associated maturation arrest and hypospermatogenesis. A power regression curve correlating the number of elongated spermatids and sperm numbers in the spermiogram was performed.

Decrease in the number of human Ap and Ad spermatogonia and in the Ap/ Ad ratio with advancing age. New data on the spermatogonial stem cell.

The numbers of Ap and Ad spermatogonia per unit section of the testis were calculated in autopsy specimens from young adults and elderly men without testicular pathology. The number of Ap spermatogonia decreased from the 6th decade of life, whereas that of Ad spermatogonia began to decrease in the 8th decade. Although it has been reported that Ad spermatogonia are more sensitive to noxious agents than Ap spermatogonia, the involution of Ap spermatogonia precedes that of Ad spermatogonia. These findings provide new information on concepts relating to the spermatogonia precedes that of Ad spermatogonia. These findings provide new information on concepts relating to the spermatogonial stem cell in man.

Liver gene expression and increase in albumin synthesis by fetal hepatocytes transplanted into analbuminemic rats.

This report describes the evolution of hepatocytes isolated from 21-day fetuses and transplanted into spleens of Nagase analbuminemic rats which have negligible serum albumin levels due to a mutation affecting albumin mRNA processing. Albumin and alpha-fetoprotein expression, in addition to other parameters related to cellular proliferation status (thymidine kinase and proliferating cell nuclear antigen expression) were studied as indicative of the behavior and evolution of the cells. In recipient rats, only a few clusters of hepatocytes could be observed in the red pulp of the spleen 24 h after transplantation. The fetal hepatocytes migrated to the liver and could be seen in portal branches immediately after transplantation. Fifteen days later, albumin mRNA was detected in recipient livers and was expressed throughout the entire 3-month study. Alpha-fetoprotein was not detected. Cell proliferation was not relevant, although 3 months after transplantation, the proliferation rates appeared to show a tendency to increase. These data demonstrate that fetal hepatocytes transplanted into spleen migrate to liver, settle there and acquire an adult phenotype free of malignant transformation. Our study is a first step towards the thorough understanding of fetal hepatocyte transplantation. The next steps will involve in-depth studies of the possibilities of genetic manipulation to achieve a high degree of repopulation/expression, employing the least possible number of donor cells, and of how the cells reach the liver parenchyma, overcoming the endothelial barrier.

Optimization of the technique to isolate fetal hepatocytes, and assessment of their functionality by transplantation.

In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative less immunogenic and more resistant to both cryopreservation and ischemic injury. In the present study, we describe the method for isolation of FH and the relationship between the transplantability of FH into the spleen of analbuminemic rats and expression of albumin mRNA. Rat FH were obtained using the nonperfusion collagenase/DNase digestion method. Nagase analbuminemic rats (NAR), a strain which bears a mutation that determines the impossibility of the normal splicing of the albumin mRNA were used as recipients. The transplanted FH immediately migrated to the liver via portal vein, and anchored there. To assess the functional state of the transplanted cells, one month after transplantation, the expression of the albumin gene was studied in the liver of the recipients.

Quantification of cell types throughout the cycle of the human seminiferous epithelium and their DNA content. A new approach to the spermatogonial stem cell in man.

The numbers of each different cell type in the human seminiferous epithelium were determined throughout the 6 stages of the cycle in both semithin and ultrathin sections obtained from 15 young adult men with normal testicular histology. Up to 4 types of A spermatogonia (Ad, Ap, Al and Ac) were distinguished. In addition, the DNA nuclear content of seminiferous epithelium cells was determined on Feulgen-stained sections. Both Ad and Ap spermatogonia showed a 2c DNA content and were present in the 6 stages of the cycle, though their numbers decreased in stages III-V. Both Al and Ac spermatogonia showed a DNA content varying from 2c to 4c. Al spermatogonia were observed in stages III-V; their numbers plus those of Ad spermatogonia in these stages were similar to the numbers of Ad spermatogonia in the other stages lacking in Al spermatogonia. Ac spermatogonia appeared in stages III-VI and their numbers plus those of Ap spermatogonia in stages III-V were similar to the numbers of Ap spermatogonia in the other stages lacking in Ac spermatogonia. The results suggest that Ad spermatogonia are the stem cells. Some of them replicate their DNA; during this replication they appeared as Al spermatogonia. Al spermatogonia divide, giving rise to both Ad and Ap spermatogonia. Some Ap spermatogonia replicate their DNA; during this process they are transformed into Ac spermatogonia which divide, giving rise to B spermatogonia.

Testicular alterations in rats due to gestational and early lactational administration of lead.

A solution of lead acetate (300 mg/L) was administered via drinking water to pregnant Wistar rats from day 1 of pregnancy to delivery (Pb-treated day 0 group) or throughout gestation and early lactation (from day 1 to day 5 postnatal) (Pb-treated day 5 group). When the pups were born, four dams and their offspring in each group (control day 0, Pb-treated day 0, control day 5, and Pb-treated day 5) were sacrificed on day 0 (day 0 groups) or on day 5 (day 5 groups). Relative testicular weight and gross testicular structure were not altered by the treatment. The seminiferous tubule diameter and the number of prospermatogonia were reduced by the treatment. Determination of the n-ploidy stage of prospermatogonia indicates that these cells have more proliferative activity in Pb-treated rats than in control rats. On the other hand, the total DNA, RNA, and protein content of the testes in treated rats was significantly reduced, but the DNA: RNA ratio remained unaltered.

[Administration of growth hormone enhances the intestinal adaptive response after resection of small intestine in rats]. / La administración de hormona de crecimiento incrementa la respuesta adaptativa intestinal posterior a una resección de intestino delgado en la rata.

The aim of this study was to assess the proliferative effect of growth hormone (GH) on the remnant intestinal mucosa after small bowel resection in the rat. Three groups (n = 8/group) of adult Wistar rats were established as follows: 1) control, 2) 90% small bowel resection (SBR) and 3) 90% small bowel resection + GH 1 mg/kg-day (SBR+GH) during 7 days. Ileal samples were taken prior to resection (basal), and at sacrifice, for assessment of intestinal mucosal growth by means of morphometric (crypt and villous length) and proliferative (proliferating cell nuclear antigen, PCNA) techniques. GH administered to resected rats (SBR+GH) significantly increased the number of proliferating cells and crypt and villous length when compared to resected non-treated animals (SBR). In conclusion, in the rat, GH markedly increases the trophic action of intestinal mucosa in hyperproliferative states like massive bowel resection, enhancing remnant bowel morphologic and proliferative adaptation.

[Somatostatin and growth hormone in post-colectomy intestinal hyperplasia in rats]. / Somatostatina y hormona de crecimiento en la hiperplasia intestinal post-colectomía en la rata.

AIM: Somatostatin exerts significant effects on gastrointestinal function that may include mucosal growth regulation, probably through its action on growth hormone release. The aim of this work was to correlate somatostatin and growth hormone plasma levels and the hyperproliferative status of intestinal mucosa after colectomy. EXPERIMENTAL DESIGN: Adult Wistar rats were divided into two groups: control sham operated (n = 8) and large bowel resection (n = 8). Seven days post-colectomy, the animals were killed. Ileal mucosal samples were assayed for proliferative status (morphometry, and proliferating cell nuclear antigen labeling) and blood samples for plasmatic somatostatin and growth hormone measurement. RESULTS: A hyperproliferative status was observed with significant increases in villous length and crypt proliferating cell nuclear antigen labeling. Plasma somatostatin showed a 95% significant decrease while growth hormone levels increased significantly. CONCLUSION: The intestinal adaptation seen after colectomy is associated with lower somatostatin and higher growth hormone plasma level, possibly by regulating the intestinal adaptative process.

[Intestinal trophic effect of neurotensin after colectomy]. / Efecto trófico intestinal de la neurotensina tras colectomía.

Neurotensin is a trophic peptide for the intestinal mucosa. Intestinal resection is a well known adaptive process of mucosal growth. Our aim was to determine the effect of exogenous neurotensin administration on intestinal mucosal growth after colectomy in the rat. Two groups: colon resection (n = 15) and colon resection plus neurotensin (n = 15, 600 micrograms/kg/day, 13 days post-surgery) were studied. Intestinal growth was assessed by means of the proliferating cell nuclear antigen (PCNA) technique on the intestinal crypt. Our results showed that neurotensin increased (p < 0.0001) epithelial cell growth when compared to non treated animals. Body weight loss was found in the non treated group but not in neurotensin treated animals. In conclusion, neurotensin increases cell growth in rats with colectomy, and maintains body weight. Neurotensin may have beneficial effects in colectomized patients.

Comparative effect of growth hormone and plerocercoid growth factor in the intestinal resection in rat.

OBJECTIVE: After massive bowel resection, absorption depends on how fast the mucosal adaptation takes place. This work aims at assessing the trophic effect of growth hormone (GH) and its analogue, the plerocercoid growth factor (PGF), on the intestinal mucosa after 90% small bowel resection. EXPERIMENTAL DESIGN: 24 male Wistar rats were divided into four groups of 6: Control (laparotomy), 90% small bowel resection (RID), resection and treatment with GH during 14 days (RID + GH) and resection and PGF treatment (RID + PGF). Intestinal mucosal adaptation was assessed by measuring mucosal weight and height, and evaluating the regenerative activity by measuring proliferation cell nuclear antigen (PCNA) labelling index. RESULTS: Bowel resection itself caused a significant increment of jejunal and ileal mucosal height in comparison with the control group. GH and PGF did not change this increase. Jejunal and ileal proliferation indexes were significantly higher than those in controls and they were significantly higher in both RID + GH and RID + PGF groups. CONCLUSIONS: GH and PGF cause a proliferative effect on the intestinal mucosa, even in hyperproliferative states such as the small bowel resection. This finding might have a clinic application.

[Importance of the colon in intestinal adaptation. Study with the proliferating cell nuclear antigen]. / Importancia del colon en la adaptación intestinal. Estudio mediante el antígeno nuclear de proliferación celular.

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein to DNA polymerase delta necessary for tissue cellular proliferation. The colon releases several peptides or hormones which are probably related to intestinal proliferation. Colonic resection determines adaptive changes in the remnant bowel. In the present study, proliferative changes after colectomy were studied by means of the murine monoclonal PC10 antibody. A control group (n = 10 rats) and a 75% proximal colon resection group (n = 10 rats) were studied. 14 days after resection, jejunal, ileal and colon samples were taken and assayed for PCNA. Relationship between immunostained nuclei and the total number of nuclei was determined. The three intestinal segments showed statistically significant increases (p < 0.001) in the number of immunostained nuclei. PCNA proliferative index was greater in the remnant large bowel.

[Effects of neurotensin on the development of suckling rats with intestinal resection]. / Efectos de la neurotensina en el desarrollo de la rata lactante con resección intestinal.

Massive intestinal resection produces malabsorption which, in the suckling rat, reduces growth. Our aim was to determine whether the proliferative action of neurotensin, can reduce the negative effects on growth induced by bowel resection. Fifteen days old suckling Wistar rats were used. Twenty rats underwent 90% midgut resection and twelve were used as controls. Half the animals were treated with neurotensin (600 micrograms/kg-day) until sacrifice 30 days later. Body and bone weight were measured and mucosal samples obtained. All resected animals lost body weight and bone weight. Neurotensin treatment reduced femur weight loss. After bowel resection, significant trophic effects were observed at mucosal level (crypt and villous size) but only in the jejunum of resected animals neurotensin treatment had a trophic effect. In conclusion, neurotensin favors intestinal adaptation after resection without improving mid-term growth in the suckling rat.

[Early or late orchidopexy? An evaluation of germ cell proliferation by PCNA immune expression]. / Orquidopexia precoz o tardía? Valoración de la proliferación de las células germinales mediante inmunoexpresión de PCNA.

A immunocytochemical study for detection of proliferating cell nuclear antigen (PCNA) in order to quantify the number of PCNA-positive spermatogonia, and cytophotometric determination of spermatogonial DNA were performed in cryptorchid and control testes. The number of PCNA-positive spermatogonia, and the average DNA content of spermatogonia in the cryptorchid testes were altered from first years of age. These precocious spermatogonial alterations suggest that the early surgical testicular descent doesn't prevent lesions of germ cells.

Carcinoma in situ of the testis in infertile men. A histological, immunocytochemical, and cytophotometric study of DNA content.

Of 723 infertile men (128 with a history of cryptorchidism) whose testes were biopsied at the outer lateral face of the testis, five presented carcinoma in situ (CIS) in one testis. These testes were removed, serially sectioned, and examined by light microscopy. In order to evaluate whether only one or two biopsies are sufficient to diagnose CIS, before sectioning the testes four biopsies were taken at the anterior face, posterior face, superior pole, and inferior pole of the testis, respectively. Two of the five men had undergone orchiopexy in infancy and the testis contained tubules with Sertoli cells and isolated spermatogonia. CIS was also present in some tubules that were principally located near the rete testis. Of the four simulated biopsies, only that performed at the posterior face of the testis revealed CIS. The other three infertile men showed tubules with complete, although reduced, spermatogenesis, and tubules lined by Sertoli cells only. CIS was found in both types of tubules. These tubules with CIS formed lobules that extended throughout the testicular parenchyma. Most simulated biopsies performed in these three testes showed CIS. The average nuclear DNA content of CIS cells was about 4c in all testes. This content was similar both in tubules with complete spermatogenesis and in tubules with Sertoli cells only.

Multinucleate spermatids in aging human testes.

A comparative morphologic study between the testes of 25 young adult men and 41 elderly men without testicular or related pathological conditions revealed the presence of multinucleate spermatids, showing up to 86 nuclei, in the testes of 3 of the elderly men. The formation of multinucleate spermatids is probably due to cell fusion, since the number of nuclei in these cells is not always 2n and multinucleate spermatocytes were uncommon. This anomaly seems to be another manifestation of an involutive process that also affects other cell types in the aging testis.