Effect of androgens on the cranial suspensory ligament and ovarian position.

Androgens have been postulated to have a major role in testicular descent via regression of the cranial suspensory ligament, which in normal rodents anchors the ovary to the retroperitoneum near the lower pole of the kidney. This study aimed to quantitate the degree of descent of the foetal ovary in androgen-treated female mice to determine the role of androgens in regression of the cranial suspensory ligament and descent of the testis. Time-pregnant mice were injected with testosterone propionate or methyl testosterone (2.5-3.0 mg) in vehicle on day 13 or 14. Control animals received vehicle only. Newborn mice were anaesthetised and dissected for macroscopic anatomy of the ovary, which was quantified by measuring the vertical distance from the lower pole of the kidney to the lower pole of the ovary. Histological analysis was also performed. The external genitalia were masculinised in all females exposed to prenatal androgens. The ovaries of treated animals were mobile, with no cranial suspensory ligament, and located slightly caudal to the kidney. Wolffian duct structures were identifiable, but the gubernaculum was qualitatively unchanged compared with control females. The ovary was displaced caudally (P< 0.001), but only 15-25% of the distance to the lower abdomen. Exogenous androgens induce regression of the cranial suspensory ligament, causing the ovary to be more mobile and lower in the abdominal cavity. However, since the testicular position at birth is at or below the bladder neck, androgen-mediated regression of the cranial suspensory ligament is only an adjunct to the control of transabdominal testicular descent.     

Androgen action during male sex differentiation includes suppression of cranial suspensory ligament development

The cranial suspensory ligament is located on the border of the cranial (mesonephric) mesentery in adult female mammals, which runs between the cranial pole of the internal genitalia and the dorsal abdominal wall. Absence of the cranial suspensory ligament in male mammals depends upon exposure of its primordium to fetal testicular androgens and is a prerequisite for testis descent. Female rats were exposed to 5alpha- dihydrotestosterone propionate at different stages of genital development, and cranial suspensory ligament development was studied in neonatal and in adult animals. Androgens suppressed cranial suspensory ligament development when exposure started during the early stages of genital development, until day 19 postconception (pc). Androgen receptor expression was immunohistochemically detected in the cranial mesentery of both sexes from day 16 pc onwards. A decrease of androgen receptor expression in female fetuses from day 18 pc onwards coincided with the appearance of a differentiated cranial suspensory ligament, as evidenced by the expression of two cell differentiation markers: alpha- smooth muscle (alpha-SM) actin and desmin. alpha-SM actin was located on the outer border of the cranial mesentery of both sexes at day 17 pc, and expression increased only in female fetuses. On day 19 pc, desmin expression was also detectable in the a-SM actin-positive cells. Proliferation and apoptosis indices of cells in the cranial mesentery, as analysed by 5'-bromodeoxyuridine incorporation and by detection of DNA strand breaks (TUNEL method) respectively, did not show any difference between the sexes, neither on day 17 nor on day 18 pc. Since primordial cells of the cranial suspensory ligament highly express the androgen receptor during the period of gestation when androgens can suppress cranial suspensory development, altered morphogenesis of these cells may be a direct consequence of androgen action.      

Development, structure and function of the cranial suspensory ligaments of the mammalian gonads in a cross-species perspective; their possible role in effecting disturbed testicular descent

The present review aims to present a perspectiveon a relatively unknown part of the mammalian internal genitalia: their cranial suspensory apparatus.This apparatus shows wide divergence of development when examined during the fetal period or during adulthood, in males or females, or in individuals across a variety of species. In rats and other mamalian species the apparatus undergoes a distinct patern of sexually dimorphic development and fetal testicular androgens are proposed to play a key role in this process. Extensive development of this suspensory apparatus in females is argued to be a part of the anatomical adaptations of the genital apparatus to support the internal genitalia throughout pregnancy, including the relatively enormous growth towards the time of parturition. Minor development of this apparatus in males is judged to be a part of the anatomical requirements allowing developing testes to become displaced from the dorsal abdominal wall during the first stage of testicular descent. Extensive development of this suspensory apparatus in males generally seems to hinder testicular descent. Accordingly, the apparatus is well developed in so-called testicond species which do not show testis descent as a part of their normal male genital development. Furthermore, arguments are adduced that inappropriate and extensive development in species with testis descent may be a key aetiological factor in the disturbance of this process. Diagnosis and treatment of human cryptorchidism might profit from including an analysis of the development and function of (remnants of) the cranial testicular and epididymal suspensory apparatus.     

Familial male pseudohermaphroditism and testicular descent in the racoon dog (Nyctereutes)

Sexual differentiation was investigated in familial male pseudohermaphroditism in Nyctereutes procyonoides (Canidae). In intersex males, development of external genital organs and prostate glandular tissue was severely disturbed; Wolffian (mesonephric) duct derivatives developed prepubertally but were absent in some adults. Müllerian (paramesonephric) duct regression was complete. Testicular descent was undisturbed. Male/female sex differences in plasma testosterone, 5 alpha-dihydrotestosterone, and luteinizing hormone concentrations were present. Intersex plasma hormone concentrations were within the normal male range. The concentration of androgen receptors in pubic skin was similar in male, female, and intersex animals and no significant differences in affinity for the ligand were detected. It was concluded that in intersex animals androgen-dependent virilisation was deficient despite the presence of androgens and androgen receptors and that this condition had not affected gubernaculum development and testicular descent.     

Biochemical and endocrine heterogeneity in the complete form of testicular feminization syndrome

A familial variant of male pseudohermaphroditism different from the classical form of the complete testicular feminization syndrome was studied. The three affected 46, XY sibling (16,18 and 20 years old) exhibited female phenotype identical to that of a 17 years old patient with the classical form, included as a control. The major endocrine and biochemical differences observed in this family, as compared with the classical form, includes: a. Markedly elevated serum levels of LH and FSH; b. Non-elevated serum testosterone levels; c. Poor testicular hCG responsiveness; d. Abnormally elevated baseline and hCG-stimulated androstenedione: testosterone ratio; e. Slight pituitary responsiveness to androgens; f. presence of residual androgen uptake by cultured fibroblasts derived from genital skin. These differences were more evident in the two older patients. All subjects presented a lack of nitrogen retention following testosterone administration. These results were interpreted as demonstrating a testicular impairment of testosterone biosynthesis in the older subjects of this family, which resulted in an unusual gonadotropin profile. The altered androstenedione: testosterone ratio suggests a secondary partially decreased activity of testicular 17-hidroxysteroid dehydrogenase, as demonstrated in TFM mice and rats. The overall data indicate an age-dependent variability in the expression of androgen insensitivity in this family, thus demonstrating the wide biochemical heterogeneity of the androgen resistant syndromes.     

Isoflurane preconditioning neuroprotection in experimental focal stroke is androgen-dependent in male mice

Isoflurane preconditioning neuroprotection in experimental stroke is male-specific. The role of androgens in the ischemic sensitivity of isoflurane preconditioned male brain and whether androgen effects are androgen receptor dependent were assessed. Male C57BL/6 mice were implanted with flutamide (androgen receptor antagonist), or castrated and implanted with testosterone, dihydrotestosterone, flutamide, letrozole (aromatase inhibitor), or vehicle 7–13 days before preconditioning. P450 estrogen aromatase wild-type and knockout mice were also evaluated. All mice were preconditioned for 4 h with 0% (sham preconditioning) or 1% isoflurane (isoflurane preconditioning) and recovered for 24 h. Mice then underwent 2 h of middle cerebral artery occlusion and were evaluated 22 h later for infarct volume. For neurobehavioral outcomes, sham and isoflurane preconditioned castrated male±dihydrotestosterone groups underwent 1 h of middle cerebral artery occlusion followed by 9 days of reperfusion. Isoflurane preconditioning neuroprotection relative to infarct volume outcomes were testosterone and dihydrotestosterone dose-specific and androgen receptor-dependent. Relative to long-term neurobehavioral outcomes, front paw sensorimotor function improved in isoflurane preconditioned mice regardless of androgen status while androgen replacement independently improved sensorimotor function. In contrast, isoflurane preconditioning improved cognitive function in castrates lacking endogenous androgens, but this improvement was absent in androgen replaced mice. Our findings suggest that androgen availability during isoflurane preconditioning may influence infarct volume and neurobehavioral outcomes in male mice following experimental stroke.     

Studies on the neuroendocrine,somatic and behavioral effectiveness of testosterone and its 5α reduced metabolites in Swiss-Webster mice

In this series of studies we examined the relative potency of testosterone and three of its 5α reduced metabolites using Swiss-Webster mice. Dihydrotestosterone and 3α-androstanediol were found to be more potent in feedback suppression of gonadotropin secretion than testosterone and 3β-androstanediol as assessed by inhibition of ovarian compensatory hypertrophy and by production of testicular atrophy. All four of these androgens were potent stimulators of seminal vesicle growth with dihydrotestosterone > 3α-androstanedione > 3β-androstanediol > testosterone. When dissolved in propylene glycol vehicle testosterone was by far the most effective stimulator of male sexual behavior, followed by 3β-androstanediol and finally 3α-androstanediol. Dihydrotestosterone failed to stimulate sexual behavior when dissolved in this vehicle; however, when dissolved in an oily vehicle dihydrotestosterone was nearly as potent as testosterone.     

Androgenic regulation of ventral epithelial bud number and pattern in mouse urogenital sinus

The ventral urogenital sinus (UGS) of control male mice has two rows of 3–4 prostatic buds at birth, but how androgens regulate ventral bud (VB) number and patterning is unclear. VBs in both sexes appeared to be a mixture of prostatic and urethral buds. UGSs from Tfm male and antiandrogen (flutamide)‐exposed mice had small VBs, suggesting that initiation of some VBs is androgen independent. Tfm male mice are widely considered completely androgen insensitive yet their UGSs were 5α‐dihydrotestosterone (DHT)‐ responsive. VBs (6–8) were generally distributed bimodally on the left‐right axis at both minimal and normal male androgen signaling. Yet control females and DHT‐exposed Tfm males had 13–14 VBs, whose left‐right distribution was fairly uniform. These results suggest that VB number and distribution respond biphasically as androgen signaling increases from minimal, and that androgens regulate bud specification. Complete VB agenesis by the selective budding inhibitor 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) required high androgen signaling. Developmental Dynamics 239:373–385, 2010. © 2009 Wiley‐Liss, Inc.     

Comparison between testosterone oenanthate-induced azoospermia and oligozoospermia in a male contraceptive study. IV. Suppression of endogenous testicular and adrenal androgens

Administration of supraphysiological doses of testosterone to normal men causes inhibition of spermatogenesis, but while most become azoospermic, 30-55% maintain a low rate of spermatogenesis. We have investigated whether there are differences in endogenous androgen production, of testicular and adrenal origin, which may be related to the degree of suppression of spermatogenesis. Thirty-three healthy Caucasian men were given weekly i.m. injections of 200 mg testosterone oenanthate (TE), 18 became azoospermic, while 15 remained oligozoospermic. Urinary excretion of epitestosterone, a specific testicular product, was reduced to <10% of pretreatment values, with no differences between the groups. Similar results were obtained for other markers of testicular steroidogenesis. Urinary and plasma adrenal androgens were also reduced during TE treatment: a statistically significant decrease in both (P < 0.001 and P < 0.05 respectively) was seen in the azoospermic but not oligozoospermic responders. These results suggest that testicular steroidogenesis is decreased to <10% by the administration of supraphysiological doses of exogenous testosterone. Differences in the degree of ongoing steroidogenesis in the testis do not appear to account for incomplete suppression of spermatogenesis, thus differences in androgen metabolism may underlie this heterogeneous response. A small but significant reduction in secretion of adrenal androgens was also detectable, the relevance of which is unclear.      

Dog kidney: Anatomical relationships between intrarenal arteries and kidney collecting system

The detailed findings of canine intrarenal anatomy (collecting system and arteries) are presented. Ninety‐five three‐dimensional endocasts of the kidney collecting system together with the intrarenal arteries were prepared using standard injection–corrosion techniques and were studied. A single renal artery was observed in 88.4% of the casts. The renal artery divided into a dorsal and a ventral branch. Using the branching pattern of the ventral and dorsal divisions of the renal artery, the vessels were classified in type I or type II. Type I presented a cranial and a caudal artery, whereas type II presented a mesorenal and a caudal artery. Cranial branches of dorsal and ventral arteries supplied the cranial pole in 90.5% of the specimens. Caudal branches of the dorsal and the ventral divisions of the renal artery irrigated both the caudal pole and the mid‐zone of the kidney in 95.8% and 98.9% of the cases, respectively. In all casts, caudal branches of both dorsal and ventral arteries supplied the caudal pole. Therefore, the caudal branches of the ventral and dorsal divisions of the renal artery are of utmost importance in the kidney arterial supply. Although many results of renal and intrarenal anatomy in dogs may not be completely transposed to humans, the anatomical relationship between arteries and the collecting system in the cranial pole of the dog kidney is similar to those in man. This fact supports the use of the dog as an animal model for urologic procedures at the cranial pole. Anat Rec, 290:1017–1022, 2007. © 2007 Wiley‐Liss, Inc.     

Androgen excess in women – A health hazard?

A significant body of evidence suggests that androgens in women may play a role in the genesis of central adiposity and type 2 diabetes. There are two principal sources of circulating androgens in females: the ovary and the adrenal gland. In hyperandrogenic women, there are elevated serum concentrations of androstenedione and testosterone and, in up to 50% of the women, dehydroepiandrosterone sulfate (DHEAS). The androgen precursor DHEAS is of exclusive adrenal origin, suggesting that hyperandrogenic women have an elevated proportion of adrenal androgen production and secretion. Another cause of androgen excess in reproductive-age women is a decreased conversion of testosterone to estradiol by the aromatase enzyme complex. In this review, we will discuss the hypothesized clinical sequel of elevated androgens in women - an aspect of women's health highly neglected. Furthermore, an attempt is made to appreciate what causes the androgens to initially rise from normal levels, allowing the onset of pathophysiological processes towards diseases.     

Partial denervation of the ovaries by transection of the suspensory ligament does not inhibit ovulation in rats treated with pregnant mare serum gonadotropin

Adrenergic nerves reach the ovary via two routes: along the arteries to the ovary and via the suspensory ligament. Results from earlier investigations suggest that denervation of the nerves along the arteries does not influence the ovulatory process. In the present study we have examined whether denervation by transection of the ovarian suspensory ligament influences the ovulatory process. Partial denervation of the ovary by transection of the ovarian suspensory ligament, sham operation, or only anesthesia were performed on immature 25-day-old rats. To induce ovulation, pregnant mare serum gonadotropin (PMSG) was injected in the morning (0800-0930), when the rats were 26 days old. This PMSG treatment normally induces ovulation around 0200 in the early morning of day 29 with subsequent formation of corpora lutea. Rats were killed 5-8 hr, 3 days, and 5 days after this ovulation time. Ovarian interstitial norepinephrine levels were markedly decreased after transection of the suspensory ligament. Ovulations had occurred in all denervated, as well as sham-operated, and control rats. The various groups did not differ in the number of ovulations per rat. Thus, the adrenergic nerves in the suspensory ligament appear not to be necessary for ovulation. Whether catecholamines themselves play a role in the ovulatory process cannot be elucidated from this experiment, since the norepinephrine content in the ovary was not totally depleted. It seems unlikely that adrenergic nerves reach the corpus luteum via the suspensory ligament, since transection of this structure did not change the luteal content of norepinephrine.     

Correlation between computed tomography and gross anatomy of the suspensory ligament of the ovary

To assess the morphology of the suspensory ligament of the ovary on CT scan examination, an anatomic study of the suspensory ligament of the ovary was performed to demonstrate its morphology on CT and to facilitate the visualization of the ovaries. Twelve female cadavers were studied after intravascular injection of latex (venous 2, arterial 6, arterio-venous 4). We first observed all the pelvises after their transection. Six dissections were then made to obtain optimal anatomic correlations on the content and relations of the infundibulo-pelvic ligament. The arterial opacifications were poor compared with to the good quality of the venous or arterio-venous opacifications. An upright correlation between CT slices and the anatomic study was made. This radio-anatomic study emphazises the importance of the veins, which really support the suspensory ligament of the ovary, as opposed to the involution of the arteries. This study points out the link between the infundibulo-pelvic ligament above and the utero-ovarian below, then with the superficial uterine vv. All these structures were well analyzed on CT and during the anatomic studies. A very well developed periovarian venous plexus which frequently concealed the ovaries was demonstrated. Some anatomic variations which were invisible on the CT scan examinations were displayed. The visualization of the suspensory ligament of the ovary should facilitate the visualization of the ovaries and could optimize the search for lymphadenopathies originating from ovarian cancers.     

Poly (ADP-ribose) polymerase-1 initiated neuronal cell death pathway—do androgens matter?

Activation of poly (ADP-ribose) polymerases (PARP) contributes to ischemic damage by causing neuronal nicotinamide adenine dinucleotide (NAD⁺) depletion, release of apoptosis-inducing factor and consequent caspase-independent cell death. PARP-mediated cell death is sexually dimorphic, participating in ischemic damage in the male brain, but not the female brain. We tested the hypothesis that androgen signaling is required for this male-specific neuronal cell death pathway. We observed smaller damage following focal cerebral ischemia (MCAO) in male PARP-1 knockout mice compared to wild type (WT) as well as decreased damage in male mice treated with the PARP inhibitor PJ34. Protection from ischemic damage provided by PJ-34 in WT mice is lost after removal of testicular androgens (CAST) and rescued by androgen replacement. CAST PARP-1 KO mice exhibit increased damage compared to intact male KO mice, an effect reversed by androgen replacement in an androgen receptor-dependent manner. Lastly, we observed that ischemia causes an increase in PARP-1 expression that is diminished in the absence of testicular androgens. Our data indicate that PARP-mediated neuronal cell death in the male brain requires intact androgen-androgen receptor signaling.     

The role of insulin 3, testosterone,Müllerian inhibiting substance and relaxin in rat gubernacular growth

Transabdominal testicular descent is influenced by various anatomical and hormonal factors and is mediated by gubernacular enlargement and regression of the cranial suspensory ligament, but its mechanism remains controversial. The aim of this study was to determine which hormones have a direct effect on the proliferation of cells in the day 17 fetal rat gubernaculum in vitro, using an organ culture system. The effects of synthetic rat insulin 3 (INSL3), inactive INSL3, dihydrotestosterone (DHT), DHT+INSL3, human Müllerian inhibiting substance (hMIS), hMIS+INSL3 and human gene 2 relaxin were tested, together with co-culture with fetal rat testis. Cell proliferation was assessed using a bromodeoxyuridine labelling index. The results showed that MIS and relaxin have a mild effect on gubernacular growth, whilst INSL3 and DHT have a more marked effect. The combination of MIS+INSL3 showed an effect close to that of co-culture with testis. However, the most pronounced effect was caused by DHT+INSL3. RT-PCR analysis indicated that the fetal rat gubernaculum strongly expresses putative INSL3 receptors, weakly expresses MIS type II receptors and does not express relaxin receptors. In conclusion, a number of different hormones directly influence growth of the gubernaculum in vitro, including the recently reported hormone INSL3. INSL3 shows a direct stimulatory effect on the swelling reaction, while DHT and MIS may have roles in augmenting this growth.     

The origin of the extrinsic adrenergic innervation to the rat ovary

Cutting the suspensory ligament reduced the ovarian content of norepinephrine (NE) to less than half that of controls and only a few blood vessels had perivascular fibers and an occasional nerve remained in the interstitial gland. Cutting the ovarian plexus had a less drastic, but similar effect on the ovarian content of NE and on the pattern of ovarian adrenergic nerves. Cutting both the suspensory ligament and ovarian plexus eliminated visualization of ovarian adrenergic nerves, but some ovarian NE was still measurable. Fluorescence and electron microscopic studies of the suspensory liagament revealed a large adrenergic nerve embedded in smooth muscle of the ligament. The nerve was also acetylcholinesterase-positive. Cutting the celiac plexus or incising a small nerve lateral to the plexus and medial to the origin of the suspensory ligament, had the same effect on the ovarian adrenergic nerves as cutting the suspensory ligament. It is concluded that the extrinsic adrenergic nerves to the rat ovary reach the organ by two routes: one via the nerve in the suspensory ligament (superior ovarian nerve), and one via the traditionally described ovarian plexus along the ovarian artery.     

Depression-like behavior of aged male and female mice is ameliorated with administration of testosterone or its metabolites

There may be a role of age-related decline in androgen production and/or its metabolism for late-onset depression disorders of men and women. Thus, the anti-depressant-like effects of testosterone (T) and its metabolites are of interest. Given that these androgens have disparate mechanisms of action, it is important to begin to characterize and compare their effects in an aged animal model. We hypothesized that there would be sex differences in depression behavior of aged mice and that androgens would reduce depression-like behaviors in the forced swim test. To investigate this, male and female mice (~ 24 months old) were subcutaneously administered T, or one of its 5α-reduced metabolites (dihydrotesterone-DHT, 5α-androstane,17β-diol-3α-diol), or aromatized metabolite (estradiol — E₂), or oil vehicle. Mice were administered androgens (1 mg/kg) 1 h before being tested in the forced swim test, an animal model of depression. We found that males spent more time immobile, and less time swimming, than females. Administration of T, DHT, or 3α-diol similarly reduced time spent immobile, and increased time spent struggling, of male and female mice. E₂, compared to vehicle administration, decreased time spent immobile of males and females, but increased time spent swimming of females and time spent struggling of male mice. Together, these data suggest that T and its 5α-reduced and aromatized metabolites have anti-depressant-like effects in aged male and female mice.