Luciano Debeljuk

Spermatogenesis in Bclw-Deficient Mice

Abstract Bclw is a death-protecting member of the Bcl2 family of apoptosis-regulating proteins. Mice that are mutant for Bclw display progressive and nearly complete testicular degeneration. We performed a morphometric evaluation of testicular histopathology in Bclw-deficient male mice between 9 days postnatal (p9) through 1 yr of age. Germ cell loss began by p22, with only few germ cells remaining beyond 7 mo of age. A complete block to elongated spermatid development at step 13 occurred during the first wave of spermatogenesis, whereas other types of germ cells were lost sporadically. Depletion of Sertoli cells commenced between p20 and p23 and continued until 1 yr of age, when few, if any, Sertoli cells remained. Mitochondria appeared to be swollen and the cytoplasm dense by electron microscopy, but degenerating Bclw-deficient Sertoli cells failed to display classical features of apoptosis, such as chromatin condensation and nuclear fragmentation. Macrophages entered seminiferous tubules and formed foreign-body giant cells that engulfed and phagocytosed the degenerated Sertoli cells. Leydig cell hyperplasia was evident between 3 and 5 mo of age. However, beginning at 7 mo of age, Leydig cells underwent apoptosis, with dead cells being phagocytosed by macrophages. The aforementioned cell losses culminated in a testis-containing vasculature, intertubular phagocytic cells, and peritubular cell “ghosts.” An RNA in situ hybridization study indicates that Bclw is expressed in Sertoli cells in the adult mouse testis. Consequently, the diploid germ cell death may be an indirect effect of defective Sertoli cell function. Western analysis was used to confirm that Bclw is not expressed in spermatids; thus, loss of this cell type most likely results from defective Sertoli cell function. Because Bclw does not appear to be expressed in Leydig cells, loss of Leydig cells in Bclw-deficient mice may result from depletion of Sertoli cells. Bclw-deficient mice serve as a unique model to study homeostasis of cell populations in the testis.

Modulation of the hypothalamo-pituitary-gonadal axis and the pineal gland by neurokinin A, neuropeptide K and neuropeptide γ

Modulation of the hypothalamo-pituitary-gonadal axis and the pineal gland by neurokinin A, neuropeptide K, and neuropeptide gamma. PEPTIDES 1999. Neurokinin A (NKA), neuropeptide K (NPK) and neuropeptide gamma (NPG) are members of the family of tachykinins, and act preferentially on NK-2 tachykinin receptors. These peptides are widely distributed and are potent stimulators of smooth muscle contraction, especially in the respiratory and gastrointestinal tract. They also induce vasodilatation and plasma extravasation. Through their effects on the vascular tone, they are also potential regulators of the blood flow and therefore of the function of many organs and tissues. Tachykinins have been demonstrated to influence the secretory activity of endocrine cells, and they may have a physiological role as regulators of endocrine functions. A number of reports have indicated that NPK, NKA and NPG act on the hypothalamo-pituitary gonadal axis to regulate functions related to reproduction. Therefore, we thought that, at this point, it was important to review the available evidence suggesting the role of these tachykinins on reproductive functions by effects exerted at 3 different levels of regulation: the hypothalamus, the anterior pituitary and the gonads. These 3 tachykinin peptides were reported to have effects on reproductive functions, acting on the control of the secretion of gonadotropin and prolactin at the level of the hypothalamo-pituitary axis, and on the steroid secretion by the testes and the ovaries. Acting on the hypothalamus, tachykinins, mainly NPK, were reported to inhibit LH secretion, but this effect is dependent on the presence of gonadal steroids. On the anterior pituitary gland, however, tachykinins were shown to stimulate LH and prolactin secretion, and this effect is also dependent on the presence of gonadal steroids. Tachykinin concentrations in the hypothalamus and pituitary are regulated by steroid hormones. In the hypothalamus, estrogens and testosterone increase tachykinin concentration. In the anterior pituitary gland, estradiol and thyroid hormones markedly depress tachykinin concentrations. Ovariectomy and exposure to short photoperiods significantly increase anterior pituitary tachykinins in the Siberian hamster. In the pineal gland, SP and NK-1 receptors are present and, more recently, the presence of NKA and probably also NPK was demonstrated. Castration and steroid replacement modified the content of tachykinins in the pineal gland. The removal of the superior cervical ganglia was followed by an increase in NKA content in the pineal gland. These results suggest that gonadal steroids may influence tachykinins in the pineal gland. In the gonads, tachykinins stimulated the secretory activity of Sertoli cells, but inhibited testosterone secretion by Leydig cells. There are very few reports on the role of tachykinins in the ovary, but some of them indicated that these peptides are present in some of the ovarian structures, and they may affect the secretion of ovarian steroids. Thus, NKA, NPK and NPG appear to have a modulatory role, mainly acting as paracrine factors, on the hypothalamo-pituitary-gonadal axis.

Cimetidine (Tagamet) Is a Reproductive Toxicant in Male Rats Affecting Peritubular Cells

Abstract Cimetidine (Tagamet) is a potent histaminic H2-receptor antagonist, extensively prescribed for ulcers and now available without prescription. Cimetidine is a known testicular toxicant, but its mechanism of action remains uncertain. Rats were treated i.p. with cimetidine either at 50 mg/kg or 250 mg/kg body weight for 59 days. Accessory sex organ weights, but not testis weight, were significantly reduced in the high dose treated groups. FSH levels were significantly elevated in both treated groups, but testosterone levels were unchanged. A high degree of variability characterized testis histology, with most tubules appearing normal and some tubules (15–17%) partially lacking or devoid of germ cells. Morphometry showed that although seminiferous tubule volume was not significantly changed, the volume of peritubular tissue was reduced in the high dose group. There was extensive duplication of the basal lamina, lamina densa in both apparently normal spermatogenic tubules and severely damaged tubules. Apoptotic peritubular myoid cells were also found. TUNEL labeling confirmed extensive apoptotic cell death in peritubular cells, but revealed apoptosis of vascular smooth muscle. Given that 1) peritubular myoid cell apoptosis occurs in apparently normal tubules, that 2) basal lamina disorders are found, and that 3) peritubular cells are lost from the testis, it is suggested that the primary event in cimetidine-related damage is targeted to testicular smooth muscle cells. This is the first in vivo-administered toxicant to be described that targets myoid cells, resulting in abnormal spermatogenesis.

Neurohypophyseal vasopressin in the Syrian hamster: response to short photoperiod, pinealectomy, melatonin treatment, or osmotic stimulation.

In the present study, the effect of photoperiod on vasopressin content in the pituitary neurointermediate lobe (NIL), as well as the ability of pinealectomy to prevent and melatonin to mimic the short photoperiod-induced changes in NIL vasopressin were studied in male Syrian hamsters. The ability of melatonin to modify the hyperosmotically stimulated vasopressin release was also determined. Exposure to short photoperiod (SD) for 4 or 10 weeks increased vasopressin content in the hamster NIL. In long photoperiod (LD)-exposed hamsters, pinealectomy induced a decrease in NIL vasopressin content, whereas no effect of melatonin injections on vasopressin storage in the NIL was detected. In SD-exposed animals, pineal removal failed to alter vasopressin content in the NIL. Hypertonic saline administration led to the expected decrease in vasopressin content in the NIL both in vehicle- and melatonin-treated animals. The hyperosmotically stimulated release of vasopressin was not modified by previous treatment with melatonin. The data from the present study show that, in male Syrian hamsters, exposure of animals to SD increases the vasopressin content in the posterior pituitary, but these changes appear not to be mediated by SD-induced changes in melatonin secretion. Furthermore, the exposure of animals to SD prevents the pinealectomy-induced changes in NIL vasopressin content. Melatonin does not modify the hyperosmotically stimulated vasopressin release in the male Syrian hamster.

Developmental changes of tachykinins in the hypothalamus and anterior pituitary of female Siberian hamsters from prepuberty to adulthood

The developmental changes of hypothalamic and anterior pituitary tachykinin concentrations were studied in female Siberian hamsters kept either under short (SD) or long (LD) daily photoperiods. The animals were killed between 15 and 70 days of age. Hypothalamic NKA sharply increased starting at 15 days up to 50 days in LD animals, and between 20 and 60 days in SD animals. Hypothalamic SP levels increased in a similar manner in SD animals, but in LD animals the increment was less pronounced, with increased levels from day 20 to 40, followed by a plateau. In the anterior pituitary gland, NKA concentrations in LD animals increased at 40 days of age, with only slight increases afterward, but overall the increment curve was considerably flatter than for hypothalamic NKA. In SD animals, the increase of anterior pituitary NKA was much steeper than in LD animals. However, the total content of NKA in the AP was similar in both SD and LD animals, because the AP weight was considerably higher in LD- than in SD-exposed hamsters. These results showed that photoperiod did not markedly affect the developmental changes in hypothalamic NKA. The developmental changes in anterior pituitary NKA concentrations were considerably smaller than in the hypothalamus in LD animals, but in SD animals they were much steeper. NKA concentrations in the anterior pituitary were markedly affected by the photoperiod. Concentrations of NKA in the anterior pituitary of the Siberian hamster at the age of 15 days of age were already higher than in the anterior pituitary of adult rats or Syrian hamsters.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurokinin A in the anterior pituitary of female rats: effects of ovariectomy and estradiol.

The effect of acute and chronic ovariectomy and the substitutive treatment with 17-beta estradiol and/or progesterone on anterior pituitary levels of neurokinin A (NKA) was studied in female rats. Acute ovariectomy did not result in significant changes of NKA in the anterior pituitary gland as compared with the levels in diestrous intact rats, but a single injection of 5 micrograms of estradiol in ovariectomized rats significantly decreased NKA levels in the anterior pituitary gland. Progesterone was without effect and did not modify the decrease of NKA in the anterior pituitary gland induced by estradiol. In rats examined 11 to 17 days after ovariectomy, NKA in the anterior pituitary gland was significantly higher than in diestrous intact rats. In the hypothalamus, ovariectomy resulted in decreased levels of NKA in the median eminence-arcuate nucleus. Estradiol significantly reduced NKA stores in the anterior pituitary gland but increased them in the whole hypothalamus and in the median eminence-arcuate nucleus. Thus, estradiol seems to be a powerful regulator of NKA stores in the adenohypophysis and also in the hypothalamus.

Tachykinins in the Pineal Gland: Effect of Castration and Ganglionectomy

In this investigation, the presence of NKA-immunoreactive substances was determined in pineal glands from intact, castrated and castrated, testosterone-treated male rats. The effect of environmental light, melatonin treatment and superior cervical ganglionectomy on pineal NKA-immunoreactive substances was also investigated. The results obtained show that NKA is present in measurable amounts in the rat pineal, and NPK is probably also present, Orchidectomy was followed by an increase in the content of NKA-immunoreactive substances in the pineal gland. The replacement treatment with testosterone propionate in castrated rats blocked this effect. NKA-immunoreactive substances were not significantly different quantitatively in pineals from rats killed under light or under darkness. The removal of the superior cervical ganglia was followed by a significant increase in the NKA-immunoreactive substance content in the pineal gland of male rats. These results indicate that NKA and other tachykinins are present in the pineal gland of the male rat, and they seem to be regulated by gonadal hormones and the innervation originated from the superior cervical ganglia.

Substance P variations in the hypothalamus of golden hamsters at different stages of the estrous cycle

The changes in substance P concentrations in the hypothalamus of female golden hamsters were studied at the different stages of the estrous cycle. Substance P levels in the hypothalamus of hamsters were highest during estrus and lowest during diestrus I and proestrus. The concentrations of substance P during diestrus II were not significantly different from those observed during estrus. These results show that substance P levels in the hypothalamus of female hamsters undergo significant changes during the estrous cycle.

Effects of neonatal administration of monosodium glutamate and castration on neurokinin A levels in the hypothalamus and anterior pituitary of rats

The effects of neonatal administration of monosodium glutamate (MSG) and castration on hypothalamic and anterior pituitary levels of neurokinin A (NKA) were studied in male and female rats killed at 46 days of age. In male rats treated neonatally with MSG, body, anterior pituitary, testis, ventral prostate, and seminal vesicle weights and serum testosterone levels were significantly lower than in saline-injected controls. Hypothalamic NKA was significantly lower in MSG-treated male rats as compared with the controls, and no apparent changes were recorded in anterior pituitary NKA. Orchidectomy was followed by a significant decrease in hypothalamic NKA in saline controls, but not in MSG-treated rats. In female rats treated with MSG, there was a significant decrease in body, anterior pituitary, and ovarian weights, as compared with saline-injected controls, but no significant differences were observed in uterine weights and serum estradiol levels. Hypothalamic NKA was lower, although not significantly, in MSG-treated rats as compared with the respective controls, and no differences were recorded in anterior pituitary NKA levels. Ovariectomy was followed by a significant decrease in hypothalamic NKA in both MSG-treated and control rats, but NKA in the anterior pituitary was significantly increased after ovariectomy only in saline-treated controls, whereas MSG-treated females failed to show this response. It is concluded that neonatal MSG treatment resulted in a decrease of hypothalamic NKA, which was particularly pronounced in male rats without any significant change in anterior pituitary NKA levels. The response of hypothalamic NKA to castration and the response of anterior pituitary NKA to ovariectomy were also altered in MSG-treated rats; this may reflect a functional block of some neuroendocrine functions of the hypothalamus that resulted from the neuronal lesions induced by MSG.

Effects of overexpression of growth hormone-releasing hormone on the hypothalamo-pituitary-gonadal function in the mouse.

In this investigation, the neuroendocrine alterations induced by high, chronic circulating levels of endogenous growth hormone (GH) were studied in transgenic mice with ectopic overexpression of the human growth hormone-releasing hormone (h-GH-RH) gene. In comparison with their normal littermates, transgenic h-GH-RH mice had elevated plasma levels of GH, prolactin (PRL), and corticosterone. In addition, they had elevated body, liver, kidney, spleen, and pituitary weights compared with normal mice. Testis and seminal vesicle weights were also increased in transgenic mice. Although basal plasma luteinizing hormone (LH) levels, plasma estradiol levels in females, and plasma testosterone levels in males did not differ significantly between normal and transgenic animals, the LH response to castration was severely impaired in transgenic mice of both sexes. Among the biogenic amines studied in the hypothalamus, only dopamine concentrations were significantly lower in transgenic animals compared with their normal littermates. This decrease in hypothalamic dopamine may be related to the hyperprolactinemia in transgenic animals. In vitro, pituitaries from transgenic mice released significantly higher amounts of GH, and although the basal release of LH was not different in both normal and transgenic mice, the response to gonadotropin-releasing hormone was significantly smaller in transgenic mice. Cultured anterior pituitary cells from transgenic mice secreted high quantities of GH and PRL in vitro, but these quantities significantly decreased from 1 to 8 wk in culture. These results show that high, persistent levels of circulating endogenous GH induce alterations in neuroendocrine functions related to the hypothalamopituitary-gonadal and the hypothalamo-pituitary-adrenal axes.