Lisa M. Watts

DOES TESTOSTERONE DIFFUSE DOWN THE WOLFFIAN DUCT DURING SEXUAL DIFFERENTIATION

PURPOSE Sexual differentiation in gonadal dysgenesis is commonly asymmetrical. In patients with true hermaphroditism there may be an ovary and müllerian duct on 1 side, and a testis and wolffian duct on the other side. Such asymmetry suggests that testicular hormones only act locally at this early stage of sexual differentiation. We tested the hypothesis that testosterone reaches the wolffian duct by transport down the duct rather than by simple diffusion. MATERIALS AND METHODS Mouse 14-day urogenital ridges were placed in organ culture and microinjected with testosterone-albumin-fluorescein isothiocyanate. RESULTS At 17 hours fluorescence was found throughout the wolffian duct and by 48 hours it was maximal in the dilated caudal end. CONCLUSIONS Our results support the hypothesis that androgens may be transported along the wolffian duct. Secretion of testicular hormones into the wolffian duct may maintain hormone levels in the biologically active range.

Germ Cell Development in Neonatal Mouse Testes in Vitro Requires Müllerian Inhibiting Substance

To study the effect of müllerian inhibiting substance on testicular germ cell development, especially on gonocytes, whole testes (156) from newborn mice were cultured for 1 to 7 days in vitro. The synthetic medium contained either 10% fetal calf serum, which itself contains endogenous müllerian inhibiting substance, or transferrin, insulin and retinoic acid. Human recombinant müllerian inhibiting substance, rabbit antiserum against müllerian inhibiting substance and/or normal rabbit serum was added to some cultures. The cultured testes were fixed in Stieves fixative and stained with hematoxylin and eosin, and the numbers and types of germ cells per tubule were counted under a light microscope. Preliminary studies showed that germ cell development in newborn mouse testes was similar in vitro to that observed in vivo, except for delay in vitro. Normal germ cell maturation from gonocytes to primary spermatocytes occurred in testes cultured with 10% fetal calf serum only (i), 10% fetal calf serum plus müllerian inhibiting substance plus anti-müllerian inhibiting substance antibody (ii), 10% fetal calf serum plus normal rabbit serum (iii) and transferrin, insulin and retinoic acid plus müllerian inhibiting substance (iv). Maturation from gonocytes to A-type spermatogonia was arrested in testes cultured with 10% fetal calf serum plus anti-müllerian inhibiting substance antibody (p < 0.01), transferrin, insulin and retinoic acid alone (p < 0.001) and transferrin, insulin and retinoic acid plus müllerian inhibiting substance plus anti-müllerian inhibiting substance antibody (p < 0.001). The results are consistent with the hypothesis that müllerian inhibiting substance may be involved in postnatal gonocyte development and suggest that it may be useful to treat infertility associated with undescended testes.

In vitro fusion of human inguinal hernia with associated epithelial transformation.

The processus vaginalis (PV) is a peritoneal diverticulum which forms to allow descent of the fetal testis to the scrotum. During human development fusion and obliteration of the PV often fails to occur with the result that inguinal hernias are the most prevalent congenital abnormality requiring surgery in childhood. Androgen is proposed to regulate testicular descent via the genitofemoral nerve which releases the neuropeptide calcitonin gene-related peptide (CGRP). It is possible that subsequent fusion of the PV and tissue remodelling following descent is indirectly controlled by androgen via CGRP action. An organ culture assay was developed to assess fusion of the PV taken from inguinal herniotomy in infants. Fusion was induced in vitro by CGRP but not by CGRP 8–37, CGRP 27–37 or dihydrotestosterone in equimolar concentrations. Fusion was accompanied by transformation of the epithelium, as shown by staining of intermediate filament proteins, cytokeratin and vimentin. Localization studies for CGRP receptors on 25 specimens indicated CGRP acts on mesenchymal fibroblasts but not directly on PV epithelium suggesting an indirect pathway. Hepatocyte growth factor/scatter factor was found to induce fusion of PV and may be involved as an intermediate molecule in the fusion cascade. This study represents the first approach to understanding the humoral control and underlying mechanism by which the PV fuses.

Both gonadotropin and testosterone fail to reverse estrogen-induced cryptorchidism in fetal mice: further evidence for nonandrogenic control of testicular descent in the fetus

Estradiol benzoate (E2B) causes cryptorchidism in fetal mice, with suppression of androgen secretion. Simultaneous gonadotropin (hCG) injection was reported to restore Leydig cell function and testosterone mediates testicular descent. To investigate whether hCG or testosterone fully reverses the cryptorchidism as well as testosterone secretion, fetal mice were exposed to E2B with or without hCG/testosterone. Normal transabdominal testicular descent was significantly inhibited by E2B or diethylstilbestrol injection on day 14 of gestation (n = 41; P < 0.005). Both hCG 50–100 IU (n = 59) and testosterone 5 mg (n = 45) given simultaneously with E2B failed to restore normal testicular descent (P < 0.005), with the testicular position the same as after E2B treatment alone. Mean testicular testosterone content at birth after E2B administration was 66±55 pg/testis (n = 15), while following E2B plus hCG it was 115±58 pg/testis (n = 10). Normal control mice had a testicular testosterone content of 882±422 pg/testis (n = 12). These results make the view that hCG or testosterone can reverse estrogen-induced cryptorchidism in the fetus questionable. It is proposed that estrogen not only suppresses the hypothalamic-pituitary axis and androgen secretion, but also has a direct inhibitory effect on testicular descent unrelated to androgen action. The action of müllerian inhibiting substance is known to be inhibited by estrogen during müllerian duct regression, and this may be the mechanism by which estrogen inhibits testicular descent.

Apoptotic cell death and fertility in three unilateral cryptorchid rat models.

Abstract Three rat strains have been studied, using a sensitive apoptotic detection method for germ-cell degeneration, to resolve the controversy regarding the effect of cryptorchidism on the contralateral descended testis (CDT). Sprague Dawley and Buffalo rats were made cryptorchid by operation at 20–22 days of age, while trans-scrotal (T-S) rats were a congenitally unilateral cryptorchid strain. Sham operated rats or normal T-S littermates were used as controls. Experiments were performed over a period ranging from 2 weeks to 18 months. Testis weight was assayed, as was the detection of apoptosis by agarose gel laddering and immunohistochemistry by using the TUNEL method. Labeled cells in 150 cross-sectioned testis tubules were counted on the TUNEL stained slides and the mean number of labeled cells per tubule was calculated. Paternity studies on Sprague Dawley and T-S rats were carried out at 12 and 24 weeks of age to assess fertility by the resultant number of pregnancies and litter sizes. Both Sprague Dawley and T-S rat models showed a biphasic distribution of apoptosis levels. This biphasic distribution was not observed in Buffalo rats as they were only studied at later time points (12–20 weeks). A significant effect on either testis weight or apoptosis in the CDT compared with the control descended testis (P ≥ 0.1) has not been found in these three cryptorchid models, and the present results are discussed with reference to observations of other researchers in rodents and humans. While the cryptorchid testis showed a high level of labeled apoptotic cells per tubule in all rat strains, fertility was not affected and remained the same as controls at 12 and 24 weeks. There was, however, a marked strain difference in fertility in T-S as compared with Sprague Dawley rats. After 24 weeks of cryptorchidism, both control and cryptorchid T-S rats had a 44% pregnancy incidence compared with a 90% pregnancy incidence in Sprague Dawley rats. In addition, litter size in T-S control and cryptorchid rats were small compared with those of Sprague Dawley rats at 12 and 24 weeks.

Localization of calcitonin gene-related peptide within the genitofemoral nerve in immature rats

BACKGROUND/PURPOSE Calcitonin gene-related peptide (CGRP) has been proposed to influence migration and testicular descent by release from the genitofemoral nerve. The site of CGRP within the nerve has been controversial, with conflicting views on whether CGRP is synthesised and released from the motor nerves. METHODS The genitofemoral nerve (GFN) was retrogradely labelled by fluorescent dye (DAPI) in 25 Sprague-Dawley rats (days 5, 16, and 31, n = 8 in each group; day 35, n = 1). Spinal cords and dorsal root ganglia (DRG) were removed two to three days later and sectioned for immunofluorescence. Substance P and CGRP-containing cells were labelled with fluorescein-linked antibodies. Specimens were examined by double fluorescence to identify cells with both markers. RESULTS The motor nucleus of the GFN contained 119 cells on day 7 and 284 cells by days 19 through 34. A prominent band of CGRP-containing fibers, arising from the dorsal horn, synapsed with the GFN motor nucleus itself. CGRP-labelled GFN cells were found in the DRG by double labelling. CONCLUSIONS CGRP from the GFN may affect gubernacular migration by release from the sensory nerves, rather than motor nerves as previously thought. The GFN motor nucleus receives CGRP-containing innervation from the dorsal horn, which may form part of the cremasteric reflex.

CONGENITAL UNDESCENDED TESTES IN NEONATAL PIGS AND THE EFFECT OF EXOGENOUS CALCITONIN GENE-RELATED PEPTIDE

PURPOSE We investigated the neonatal piglet as a possible animal model for cryptorchidism and to determine whether calcitonin gene-related peptide (CGRP), which has been proposed to regulate inguinoscrotal testicular descent, could induce testicular descent in piglets with congenital cryptorchidism. MATERIALS AND METHODS We examined 38 cryptorchid piglets to document the anatomy in 8 and to investigate the role of CGRP in 30. The 2-week-old piglets were allocated randomly to receive a mini-osmotic pump containing CGRP at various concentrations or phosphate buffered saline. The pump was inserted surgically into the ipsilateral scrotum, with the contents blinded to the surgeon. The positions of the testes, pump and anatomical landmarks were measured and photographed. The pigs were sacrificed and dissected 2 weeks later, and the positions were remeasured and photographed. The testes were examined histologically. RESULTS The 3 variants of cryptorchidism observed were intra-abdominal in 20 cases, inguinal in 9 and lateral inguinal ectopic in 9. CGRP had no effect on intra-abdominal or ectopic testes. In contrast, for inguinal testes exogenous CGRP caused a slight but significant 10 +/- 7.9 mm. descent towards the pump in 5 cases compared to -2.9 +/- 5.8 mm. in 4 controls. CONCLUSIONS Exogenous CGRP stimulated migration of inguinal testes that had been arrested in the line of descent while ectopic testes did not respond. These results support a role for CGRP in testicular descent and suggest that a slow release depot preparation might be useful as a possible treatment in some forms of cryptorchidism.

Characterization of the gubernacular contractile response to calcitonin gene-related peptide in the mouse

Calcitonin gene-related peptide (CGRP) has no effect on quiescent skeletal muscle, but cultured neonatal mice gubernacula show rhythmic contractions in response to CGRP. This study investigated whether these contractions (1) require calcium ions, (2) are activated via acetylcholine receptors, or (3) require dihydropyridine receptors. Gubernacula (n = 20 for each experiment) from male mice aged 7 days old were preincubated for 30 minutes in aerated culture medium (Krebs-Henseleit solution) with (1) up to 1.8 mmol/L of calcium with or without CGRP (714 nmol/L), (2) up to 5.0 mumol/L of curare with CGRP and calcium (1.8 mmol/L), or (3) up to 1 mumol/L of nifedipine with CGRP and calcium. Then they were placed on agar-coated grids over the same compound solutions, and observed for 2 days by video camera to see contractions. Of gubernacula cultured with calcium (1.8 mmol/L) and CGRP, 90% showed contractions, which decreased to 20% without CGRP (P < .001) and 15% without calcium (P < .001). Only 5% of gubernacula cultured without both CGRP and calcium showed contractions. Also these contractions in response to CGRP depended on calcium concentrations in a dose-dependent manner. Curare did not suppress contractions. With increasing dose of nifedipine, the percentage of gubernacula contracting decreased from 95% to 0%. These results suggest that developing mouse gubernacular contractions in response to CGRP require influx of calcium ions to the sarcoplasm via dihydropyridine receptors in a similar manner to contractions of immature cardiac or smooth muscles, and these contractions are not activated via acetylcholine receptors.