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ARTILE |
Hormone Targets, Institut National de la Santé et de la Recherche Médicale Unité 584 (N.B., P.I-B., P.A.K.), Faculté de Médecine Necker-Enfants Malades, 75015 Paris, France; and Groupe d’Etude de la Reproduction chez le Mâle-Institut National de la Santé et de la Recherche Médicale Unité 435 (N.M., C.P., H.K., A.M.T., B.J.), Université de Rennes 1, 35042 Rennes Cedex, Bretagne, France
Address all correspondence and requests for reprints to: Dr. Nadine Binart, Hormone Targets, Institut National de la Santé et de la Recherche Médicale Unité 584, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75015 Paris, France. E-mail: binart{at}necker.fr.
Abstract
Mice with a targeted disruption of the prolactin (PRL) receptor gene were used to study the physiological role of PRL in the control of the male reproductive function. Fertility parameters as well as body and reproductive organ weights (epididymis and testes) were unaffected in PRL receptor knockout mice. Testicular histology and sperm reserves were also normal. Compared with wild-type animals, knockout mice had no significant difference in basal plasma LH, FSH, and testosterone levels, and the weight of seminal vesicles and prostate was unaffected. Moreover, no alteration was detected in human chorionic gonadotropin-induced testosterone levels. It is concluded that the absence of PRL signaling is not detrimental to male testicular function and to fertility in the mouse.
WHEREAS PROLACTIN (PRL) has long been known to be the hormone responsible for mammary gland development and lactation in females, its role in the male has puzzled investigators ever since it has been shown to be present in the anterior pituitary. Initially, no clear function could be ascribed to PRL in male mammals, including humans (1, 2). However, more recent data have suggested that, generally, this hormone positively modulates several aspects of testicular function. Thus, PRL has been presented as being involved in the maintenance of cellular morphology (3) and in the up-regulation of LH receptor number on Leydig cells (4, 5). Along with LH, it has also been proposed to be implicated in the stimulation of steroidogenesis and androgen production (5, 6, 7), whereas, in contrast, it could be involved in the inhibition of aromatase activity (8). In vitro, PRL has been shown to increase FSH receptor number in Sertoli cells (9). It has also been suggested that PRL is involved in the rate of spermatocyte-spermatid conversion (3). Moreover, several in vitro effects on spermatozoa have been reported: a rise in calcium binding and/or transport of ejaculated and epididymal spermatozoa (10), an increase in energy metabolism (11), a maintenance of mobility and attachment to the oocyte (12), and a reduction in the time required to achieve capacitation (12). PRL also has metabolic effects on sex accessory organs (13, 14, 15). The effects on prostate include increased levels of androgen receptors (16, 17), involvement in estrogen-induced inflammation (18), increased epithelial secretory function (19, 20), and augmented energy metabolism. Stimulation of the level of IGF-I and its receptor has been also reported in the prostate (17).
In addition to these in vitro data, it has been shown that, in the mouse, congenital PRL deficiency caused by recessive mutations at the pit-1 locus (Snell dwarf; Ames dwarf) is associated with reduced testosterone levels, a decrease in testicular LH and PRL receptor (PRLR) number, and a severe suppression of fertility (21, 22). However, these effects may well result from a drop in circulating gonadotropin levels observed in these studies. In contrast, the model of PRL knockout (KO) mouse has allowed the demonstration that, if PRL has a physiological role in the control of LH release and in the regulation of the growth of accessory reproductive glands, it does not seem to be directly required for the maintenance of circulating testosterone and fertility (23). The development of PRLR KO mice (24) has allowed us in the present study to reinvestigate the possible involvement of PRL in testicular function. We demonstrate that none of the male reproductive tract organ parameters or functions investigated was affected in this model.
Materials and Methods
Animals and mating trials
The animals were produced by crossing animals heterozygous for the PRLR. Mice were housed under normal laboratory conditions in a 12-h light, 12-h dark cycle (0700–1900). The temperature was controlled (21 C), and the animals had free access to tap water and standard pelleted animal food. The progeny was classified by PCR analysis of DNA extracted from tails clipping as described previously (25). After reaching adulthood (2–3 months of age), each male was placed for 30 d in a cage with two virgin females, and then the females were checked daily for the presence of a vaginal plug. Mating behavior (frequency of mounting and copulation) was analyzed, and immediately after parturition the size of the litter was recorded. The local committee on animal care approved all animal protocols.
Human chorionic gonadotropin (hCG) test
Ten PRLR KO and 10 wild-type mice were chosen at random and treated with an ip injection of hCG (Organon, Puteaux, France) in PBS, at 15 IU/animal. Controls (n = 10) were injected with PBS alone. Two hours after injection, all animals were decapitated, the blood collected, and the plasma separated and stored at -20 C for hormones assays.
Collection of tissues
At the time of autopsy, testes, epididymis, ventral prostate, and seminal vesicles of the KO and wild-type mice were collected and weighed. One testis was immediately fixed in Bouin’s fluid for histology, whereas the other testis was stored at -20 C until sperm reserves were counted as previously described (26). Blood plasma was saved for measurements of LH, FSH, and testosterone by RIAs previously validated for use in mouse plasma.
The serum concentrations of LH and FSH were measured using [125I]LH, [125I]FSH, and materials obtained from the National Hormone and Pituitary Program. The lower limits of detection for these assays are 0.11 ng/ml and 1 ng for LH and FSH, respectively; values are expressed in relation to the standards. The intra- and interassay coefficients of variation were 5 and 7%, respectively. Serum testosterone levels were measured with a tritium-based RIA, with a 10–13% interassay variation. All samples were measured in the same assay.
The results were expressed as the mean ± SEM. The variance analyses using unpaired two-tailed Student’s t test were used for comparing the values measured between PRLR KO and wild-type animals.
Results
Body and reproductive organ weights
No apparent change was seen in the health status of PRLR KO mice compared with wild-type animals. This was reinforced by our data, which did not show any change in body weight of young KO animals (31.90 ± 0.43/32.80 ± 0.47 for +/+ mice). No change was seen in testis, epididymis, seminal vesicle, or prostate weights, and testis histology was normal (Fig. 1
). Moreover, sperm reserves were similar to those of controls (Fig. 2).
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Hormonal status
Pituitary hormones.
Basal plasma FSH and LH levels were not significantly changed in PRLR KO vs. wild-type males (Table 1).
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). In agreement with these observations, the seminal vesicles and prostate weights were not affected in homozygous PRLR KO, compared with wild-type males (Fig. 1
). Furthermore, the response to hCG administration was not modified in the PRLR KO animals (Fig. 3).
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In the past, a number of studies have pointed out the possible influence of PRL in the control of various aspects of male reproductive function. However, until recently, direct effects of PRL, such as the increase in Leydig cell LH receptor number (4), the enhanced sensitivity of Leydig cells to LH stimulation (5), or the stimulation of Sertoli FSH receptor number (9), could only be ascertained in vitro. Alternatively, a few studies were undertaken in vivo, but under conditions that did not permit to discrimination between the possible direct effects of PRL and the effects that could have been mediated through alterations in the hormones affected by PRL. In fact, it is well established that PRL can alter secretion of gonadotropins by the pituitary gland with subsequent changes occurring in spermatogenesis (27). A classical example of this is that, in hyperprolactinemic men, the abnormalities of spermatogenesis observed are currently thought to be a consequence of impaired testosterone levels due to LH release defects (28). The same applies to the phenotype observed in mice affected by a congenital PRL deficiency caused by recessive mutations at the pit-1 locus (22). The recent analysis of the phenotype of genetically engineered mice null for PRL gene has represented a notable breakthrough. Thus, Steger et al. (23) have recently shown that in the absence of PRL, fertility and testosterone levels are normal. The present study using PRLR KO mice totally confirms this finding. In contrast to our previous observation suggesting a delay of male fertility (24), no change of this parameter was seen in close studies. This discrepancy is explained by the use of a larger number of animals in the present mating trials and by the fact that the present study was undertaken on pure 129/Sv genetic background, instead of the mixed genetic background used previously. Furthermore, we demonstrate that the total ablation of PRLR expression has no significant consequence on spermatogenesis as assessed by testicular histology and measurement of testicular sperm reserves, fertility in term of sexual behavior and mating outcome, and androgen status, as shown by unchanged secondary accessory organ weights and basal and hCG-induced circulating testosterone. Therefore, we conclude that PRL is not a essential player in the control of male reproductive function in the mouse.
Footnotes
This work was supported in part by grants from Institut National de la Santé et de la Recherche Médicale and the Ministère de l’Education Nationale de la Recherche et de la Technologie (No. 1A010G).
Abbreviations: hCG, Human chorionic gonadotropin; KO, knockout; PRL, prolactin; PRLR, PRL receptor.
Received April 1, 2003.
Accepted for publication June 30, 2003.
References
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