Effect of Estrogen Receptor-Subtype- Speciﬁc Ligands on Fertility in Adult Male Rats

K DUMASIA and others Effect of ER selective ligands 225:3 169–180 Research on male rats

Effect of estrogen receptor-subtype- speciﬁc ligands on fertility in adult male rats

Kushaan Dumasia, Anita Kumar, Leena Kadam† and N H Balasinor Correspondence should be addressed Department of Neuroendocrinology, National Institute for Research in Reproductive Health (Indian Council of to N H Balasinor Medical Research), Parel, Mumbai 400 012, India Email †L Kadam is now at Department of Physiology, School of Medicine, Wayne State University, Detroit, Michigan, USA [email protected]

Abstract

Maintenance of normal male fertility relies on the process of spermatogenesis which is under Key Words complex endocrine control by mechanisms involving gonadotropin and steroid hormones. " estradiol Although testosterone is the primary sex steroid in males, estrogen is locally produced in the " estrogen receptor testis and plays a very crucial role in male fertility. This is evident from presence of both the " male fertility estrogen receptors alpha (ERa) and beta (ERb) in the testis and their absence, as in the case of " selective ligands knockout mice models, leads to sterility. The present study was undertaken to understand individual roles of the two ERs in spermatogenesis and their direct contribution towards the maintenance of male fertility using receptor-subtype-speciﬁc ligands. Administration of ERa and b agonists to adult male rats for 60 days results in a signiﬁcant decrease in fertility, mainly due to an increase in pre- and post-implantation loss and a concomitant decrease in

Journal of Endocrinology litter size and sperm counts. Our results indicate that ERa is mainly involved in negative feedback regulation of gonadotropin hormones, whereas both ERs are involved in regulation of prolactin and testosterone production. Histological examinations of the testis reveal that ERb could be involved in the process of spermiation since many failed spermatids were observed in stages IX–XI following ERb agonist treatment. Our results indicate that overactivation of estrogen signaling through either of its receptors can have detrimental effects on the fertility parameters and that the two ERs have both overlapping and distinct

roles in maintenance of male fertility. Journal of Endocrinology (2015) 225, 169–180

Introduction

It is well established that maintenance of normal male The importance of E2 is highlighted by the fact that E2 is fertility depends on spermatogenesis, the process of produced locally in the adult testis by most of the germ proliferation and differentiation of germ cells into mature cells, Sertoli cells and Leydig cells through the conversion

spermatids, and is under hormonal control. Although of testosterone to E2 by the enzyme aromatase cytochrome testosterone and the gonadotropin hormones follicle- P450 (Carreau et al. 1999).

stimulating hormone (FSH) and luteinizing hormone The cellular effects of E2 are mediated through its

(LH) are key players in spermatogenesis, estradiol (E2)is receptors; estrogen receptor a (ERa) and b (ERb) which now recognised to play an important role in testicular belong to the steroid hormone superfamily of nuclear physiology and spermatogenesis (Carreau et al. 2007). receptors and upon binding to their ligands act as

http://joe.endocrinology-journals.org Ñ 2015 Society for Endocrinology Published by Bioscientiﬁca Ltd. DOI: 10.1530/JOE-15-0045 Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 10:20:23AM via free access Research K DUMASIA and others Effect of ER selective ligands 225:3 170 on male rats

transcription factors and alter the rates of transcription of by both the receptors. Also, many environmental xenoes-

E2-responsive genes (Hall et al. 2001). Although both ERa trogens have different binding afﬁnities to the two ERs

and b bind to E2 with equal afﬁnity, they have different and can thus act on them differently (Kuiper et al. 1997). biological functions as is evident from their distinct Hence, there is little information on the individual roles of expression patterns and tissue distribution. In adult rat the two ERs in spermatogenesis and their direct contri- testes, ERa is expressed in Leydig cells and in pachytene bution towards the maintenance of male fertility. spermatocytes and round spermatids in the seminiferous The ER knockout models are not appropriate for

epithelium (Bois et al. 2010). On the other hand, ERb is studying the involvement of E2 in spermatogenesis in present in most cell types in the testis like the somatic adulthood as developmental defects due to absence of the Leydig cells and Sertoli cells; and germ cells like receptors in the respective knockouts cannot be excluded. spermatogonia, pachytene spermatocytes and round In studies by Oliveira et al. (2002) the complete ER spermatids (Pelt et al. 1999). In the efferent ductules, antagonist ICI 182 780 was found to impair the function- small tubules transporting sperm from the rete testis to the ing of efferent ductules, mediated by ERa, due to which caput epididymis, ERa is the predominant receptor therewasfailureofﬂuidreabsorption, resulting in subtype expressed (Hess et al. 1997). In other reproductive accumulation of ﬂuid in the testis and disruption of organs of rats, like the epididymis, both the ERs are spermatogenesis. Thus, the use of pure anti-estrogens or

detectable, whereas in prostate ERb is the predominant ERa antagonists to study the role of E2 in spermatogenesis form expressed (Sar & Welsch 2000). would not be effective due to the primary effect on efferent The importance of ERs in male fertility was further ductules. Therefore, in the present study, we investigated highlighted by the phenotypes of the knockout mice the effects of ER-subtype-speciﬁc ligands on spermatogen- generated. Complete estrogen receptor a knockout esis and fertility using an adult male rat model. The ligands (ERaKO) and estrogen receptor b knockout (ERbKO) male used in our study were 4,40,400-(4-Propyl-[1H] pyrazole- mice generated using the Cre-loxP system have been 1,3,5-triyl) (PPT), a 410-fold selective ERa agonist (Stauffer found to be infertile. Also, ERaKO testis shows atropic and et al. 2000), 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), degenerating seminiferous tubules and the epididymis was a 70-fold selective ERb agonist (Meyers et al. 2001) and 4-[2- found to be hypospermic, whereas no histopathological phenyl-5,7-bis(triﬂuoromethyl)pyrazolo[1,5-a]pyrimidin- defects in the testis and epididymis were observed in 3-yl]phenol (PHTPP), a 36-fold selective ERb antagonist ERbKO male mice despite the sterility (Antal et al. 2008, (Compton et al. 2004). Our results indicate that the two ERs Journal of Endocrinology Chen et al. 2009a). Another cause of the increased have both overlapping and distinct roles in maintenance attention to the role of estrogens in male reproduction of spermatogenesis and male fertility. can be attributed to several reports that exposure to environmental estrogens may have a detrimental effect on the reproductive health of humans and wildlife (Toppari Materials and methods et al. 1996). Declining semen quality, sperm counts and Animals other reproductive tract disorders in men in the last few decades have also been suggested to be associated with Randomly bred adult Holtzman strain male (75 days old exposure to environmental estrogens and endocrine- weighing approximately 300 g) and female (90 days old disrupting compounds (Sharpe 1993, Daston et al. 1997, weighing approximately 250 g) rats were used for the

Marques-Pinto & Carvalho 2013). The effect of E2 and present study. The animals were maintained under xenoestrogen exposure during the fetal and neonatal controlled temperature (22G1 8C) and humidity (55G stages has been extensively studied (reviewed by Delbes 5%) conditions with a 14 h light:10 h darkness cycle. The et al. (2006)) yet relatively few studies have been done animals were supplied with a diet of soy-free, in-house

to investigate the role of E2 in maintenance of male prepared rat pellets and water and allowed to feed and

fertility in adulthood. Administration of exogenous E2 to drink ad libitum. Prior approval for the use of animals was adult male rats causes a marked decrease in fertility and a obtained from the Institutional Animal Ethics Committee. concomitant suppression of the hypothalamus–pituitary– testis (HPT) axis (Rao & Chinoy 1983, Gill-Sharma et al. PPT, DPN and PHTPP treatment 2001). However, since treatment with E2 could affect both the ERs, the reduction in fertility observed after these PPT (O99%, Axon Medchem, Groningen, The Nether- treatments would be the cumulative effect brought about lands), DPN and PHTPP (O99%, Tocris Biosciences,

Bristol, UK) were dissolved completely in vehicle DMSO No: of female rats inseminated Potency Z !100 (Sigma): saline (75:25). A total of 120 male and 240 No: of female rats exposed for mating female rats were used in the study. For each drug, male rats were divided into ﬁve groups, four treatment Sample collection groups each receiving one dose of the drug and one After the mating experiments, the control and treated rats control group which received the vehicle alone. Each in all groups were weighed and killed by decapitation, in group had eight male rats. For PPT and DPN doses of order to reduce stress- and anaesthesia-induced changes in 0.05, 0.1, 0.2, 0.4 mg/kg per day and for PHTPP doses of the hormonal proﬁle, and trunk blood was collected. The 0.05, 0.1, 0.4, 0.8 mg/kg per day were used. The doses blood was allowed to coagulate at room temperature and were administered subcutaneously every day between 10 serum was separated and stored at K80 8C for hormone and 12 h for 60 days. The duration of the study was assays. Reproductive organs like testes, epididymes, chosen to cover the entire spermatogenic cycle of the prostrate, seminal vesicles, accessory glands and pituitary rat (of 54 days). The doses were selected on the basis of gland were dissected out and wet weights were recorded. previous studies reported in literature (Santollo & Eckel Paired weights were taken for testes, epididymes, seminal 2009, Campbell et al. 2010, Santollo et al. 2010, Umar vesicles and accessory glands. et al. 2011).

Epididymal sperm counts and motility Mating studies Epididymal sperm counts and motility assessment were Control and treated rats were cohabited with normal performed for all the control and treated male rats. Brieﬂy, cycling females, at a ratio of one male:two females, a both the cauda epididymides were excised and dissected week before completion of 30 and 60 days of treatment. in 10 ml of DMEM (Sigma) pH 7.4, preincubated at 37 8C

Daily vaginal smears were taken to check for the presence with 5% CO2, in a petri dish to disperse sperm from the of sperm. The occurrence of mating was conﬁrmed by tubules. The petri dish was incubated at 37 8C for 10 min thepresenceofcopulatoryplug/spermatozoainthe and sperm concentration was measured using Neubauer’s vaginal smear/persistent diestrus (O11 days). The day hemocytometer after appropriately diluting the sample. when spermatozoa were seeninvaginalsmearswas The sperm count for each group was expressed as millions designated as day 0 of gestation. In cases of persistent per cauda epididymidis. Sperm motility was analyzed Journal of Endocrinology diestrus, the last estrus before the appearance of persist- by computer-assisted semen analysis using an HTM-IVOS ence diestrus was considered as day ‘0’ of gestation. The motility analyser (Hamilton Thorne Research, Beverly, gravid females were killed between gestation days 17 and MA, USA). All the procedures were performed at 37 8C, and 19 and the uterus and ovaries were exposed. The numbers all equipment and reagents that came into contact with of live and resorbed fetuses, implantation sites (IS) and the sperm were prewarmed to and maintained at 37 8C. corpora lutea (CL) were noted. The following fertility parameters were calculated (Gill-Sharma et al.1993, Hormone assays Balasinor et al. 2001): Steroid hormones like testosterone and E2 were estimated from the serum using commercial ELISA kits from % Pre-implantation loss ðPILÞ Diagnostic Biochem Canada, Inc. (London, Ontario, No: of CL K No: of IS Canada) following the manufacturer’s instructions. Z !100 No: of CL Serum FSH, LH and prolactin were estimated on a Luminex 200IS platform using a Milliplex Map Rat % Post-implantation loss ðPOLÞ Pituitary Magnetic Bead Panel Kit (Millipore Corporation, Billerica, MA, USA) as per the manufacturer’s instructions. : K : Z No of IS No of live fetuses ! Serum samples were diluted 1:3 with serum matrix and all : 100 No of IS samples were measured in duplicate.

Litter size: the average number of live fetuses in one Histological examination of the testis litter sired by male. Potency: the ability of male rats to inseminate the The testes were dissected out and fixed in Bouin’s fixative females. It is expressed as: for 24 h. After primary fixation, each testis was cut into

three pieces (3–5 mm thick) and refixed in fresh fixative 0.05–0.2 mg/kg per day after both 30 and 60 days of PPT for 24 h. The tissues were then dehydrated through grades treatment and 60 days of DPN treatment (Fig. 1B and E). of alcohol, cleared in xylene and embedded in paraffin There was no significant change in post-implantation wax. The paraffin-embedded tissue blocks were sectioned embryo loss in females mated with PHTPP-treated males at at a thickness of 5 mm, mounted on glass slides and stained any of the doses after 30 and 60 days (Fig. 1H). with Hematoxylin and Eosin then observed under a Ziess Axioskop photomicroscope at 100! magnification. The Litter size A significant decrease in litter size was staging of seminiferous tubules was carried out according observed in females mated with males treated with PPT to the standard published criteria (Hess 1990). For and DPN at doses 0.05–0.2 mg/kg per day after both 30 and evaluation of spermiation failure, the numbers of stage 60 days of treatment (Fig. 1C and F). No significant change IX–XI tubules showing failed spermatid(s) were counted in in litter size was observed in females mated with males five cross sections for each animal (five animals per group). treated with PHTPP after 30 and 60 days treatment (Fig 1I). The percentage of tubules showing spermiation failure was calculated as a ratio of number of stage IX–XI tubules Potency A significant decrease in potency upon PPT showing failed spermatids to the total number of stage treatment with a complete loss of potency at the highest IX–XI tubules counted!100. dose of 0.4 mg/kg per day was observed (Fig. 2A). There was a significant decrease in potency at 0.1 mg/kg per day DPN and at 0.8 mg/kg per day PHTPP group. The potency Statistical analysis was unaffected by all the other doses of DPN and PHTPP Data are presented as meanGS.E.M. and were analysed (Fig. 2B and C). using GraphPad Prism Software version 5.0 (San Diego, CA, USA). The percentage pre- and post-implantation loss Effect on sperm counts and motility (PIL and POL) calculated for each of the two females mated with each male, was averaged and a total average of Caudal sperm counts were significantly reduced after all percentage PIL and POL was calculated for all the males doses of PPT treatment, whereas motility was not affected in treatment and control groups (nZ8 per group). Non- (Fig. 2D and G). Sperm counts and motility were parametric data like PIL and POL, litter size and hormone significantly decreased only at 0.05 and 0.1 mg/kg per levels were subjected to Kruskal–Wallis analysis and day DPN treatment, whereas in the groups treated with Journal of Endocrinology compared using Dunn’s multiple comparison tests. Para- higher dosages of DPN there was no effect (Fig. 2E and H). metric data on sperm counts, motility, reproductive organ No difference in sperm counts and motility was observed weights and spermiation failure were analysed by one-way after 60 days of PHTPP treatment at any of the doses as ANOVA using the Bonferroni post-test. The level of compared with the vehicle control group (Fig. 2F and I). significance was set as P%0.05.

Effect on body weight and reproductive tissue weights

Results The terminal body weights and reproductive organ weights after PPT, DPN and PHTPP treatment are Effect on fertility parameters summarized in Table 1. A decrease in terminal body Preimplantation loss A significant increase in preim- weights were observed with higher doses of PPT, whereas plantation embryo loss was observed in females mated they were not affected by DPN or PHTPP treatment. Testis with males treated with both the agonists PPT and DPN at weights were significantly decreased with the highest dose the doses of 0.05–0.2 mg/kg per day after 30 and 60 days of PPT, with no effect due to DPN and PHTPP treatment. treatment (Fig. 1A and D). In the case of PHTPP, there was The weights of epididymis were decreased after treatment an increase in preimplantation embryo loss in females with 0.2 and 0.4 mg/kg per day PPT and 0.05 and mated with males treated with doses of 0.05–0.8 mg/kg per 0.1 mg/kg per day DPN. The weights of the prostate were day after 30 and 60 day treatment (Fig. 1G). significantly reduced in response to treatment with the highest dose of 0.4 mg/kg per day PPT, and 0.8 mg/kg per Post-implantation loss A significant increase in post- day PHTPP. The weights of seminal vesicles and coagulat- implantation embryo loss was observed in females ing glands were decreased after 0.4 mg/kg per day of PPT mated with males treated with PPT and DPN at doses of treatment only. There is a dose-dependent increase in

ABC 60 50 15 Control * ** *** * 40 ** 0.05 PPT 40 *** 10 ** 0.1 PPT 30 *** 0.2 PPT ** * 20 20 * 5

10 Litter size/male Percentage PIL/male Percentage POL/male 0 0 0 30 days 60 days 30 days 60 days 30 days 60 days

DEF40 20 15 Control *** * ** * 0.05 DPN 30 15 * 0.1 DPN *** ** *** * 0.2 DPN 20 * 10 10 ** 0.4 DPN * 10 5 Litter size/male Percentage PIL/male Percentage POL/male 0 0 5 30 days 60 days 30 days 60 days 30 days 60 days

GHI25 10 15 * Control 20 8 0.05 PHTPP *** * * *** 10 0.1 PHTPP 15 ** 6 0.4 PHTPP 0.8 PHTPP 10 4 5

5 2 Litter size/male Percentage PIL/male 00Percentage POL/male 0 30 days 60 days 30 days 60 days 30 days 60 days

Figure 1 Fertility studies after PPT, DPN and PHTPP treatment. Preimplantation loss (G, H, and I) treatments. Values are meanGS.E.M. nZ8. Asterisks indicate (A, D, and G), post-implantation loss (B, E, and H) and litter size (C, F, and I) signiﬁcant differences compared with the controls (*P!0.05, **P!0.01 after 30 and 60 days PPT (A, B, and C), DPN (D, E, and F) and PHTPP and ***P!0.0001). Journal of Endocrinology

weights of pituitary after treatment with both the agonist unaffected by any of the treatments except for treatment treatment of agonists, PPT and DPN, whereas no change with the highest dose of PPT, 0.4 mg/kg per day, where a was observed upon PHTPP treatment. signiﬁcant increase was observed (Fig. 3M, N, and O).

Effect on hormonal proﬁle Effect on testicular histology

There was a significant decrease in serum FSH for all doses No gross morphological changes were observed in of PPT treatment whereas FSH levels were not affected testicular sections from animals treated with PPT and by either DPN or PHTPP treatment (Fig. 3A, B, and C). PHTPP as compared with vehicle control at any of the Similarly, serum LH was deceased by treatment with stages of spermatogenesis. All the germ cell types with higher doses of 0.2 and 0.4 mg/kg per day PPT and LH normal stage-specific cellular associations were observed levels were unchanged after DPN and PHTPP treatment for all the doses of PPT and PHTPP treatments (data not (Fig. 3D, E, and F). Serum prolactin levels were increased shown). The somatic cells like Sertoli and Leydig cells also by all doses of PPT treatment and by the treatment with appeared normal. In the case of DPN treatment, many the highest dose of DPN and contrastingly they were hook-shaped failed step 19 spermatids were observed reduced by treatment with the highest dose of PHTPP towards the basal membrane of the tubules in stages (Fig. 3G, H, and I). Serum testosterone was decreased with IX–XI (Fig. 4C); although no other histological differences increasing doses of PPT treatment. However, there was a were noted in the other stages. There was a significant significant decrease in testosterone levels only in the increase in the percentage of stage IX–XI tubules showing 0.1 mg/kg per day DPN and 0.8 mg/kg per day PHTPP spermiation failure in the 0.05 and 0.1 mg/kg per day DPN

treatment groups (Fig. 3J, K, and L). Serum E2 levels were groups as compared with the control group (Fig. 4E).

ABC 150 150 150

100 100 100 * * * * 50 50 50 Percentage potency Percentage potency Percentage potency

0 *** 0 0

Control Control Control 0.1 PPT 0.2 PPT 0.4 PPT 0.1 DPN 0.2 DPN 0.4 DPN 0.05 PPT 0.05 DPN 0.1 PHTPP0.4 PHTPP 0.8 PHTPP 0.05 PHTPP DEF 25 20 20 ) ) ) 6 6 20 6 15 15 15 * *** 10 * 10 10 * 5 5 5 No of sperm (×10 No of sperm (×10 No of sperm (×10 per cauda epididymis per cauda epididymis per cauda epididymis 0 0 0

Control Control Control 0.1 PPT 0.2 PPT 0.4 PPT 0.1 DPN 0.2 DPN 0.4 DPN 0.05 PPT 0.05 DPN 0.1 PHTPP 0.4 PHTPP0.8 PHTPP 0.05 PHTPP GHI 100 100 100

80 80 * 80

60 60 60

40 40 40

20 20 20 Percentage sperm motility Percentage sperm motility Percentage sperm motility 0 0 0

Control Control Control 0.1 PPT 0.2 PPT 0.4 PPT 0.1 DPN 0.2 DPN 0.4 DPN 0.05 PPT 0.05 DPN 0.1 PHTPP 0.4 PHTPP 0.8 PHTPP 0.05 PHTPP Journal of Endocrinology Figure 2 Decrease in percentage potency (A, B, and C), sperm counts expressed as (C, F, and I) treatment. Values are meanGS.E.M. nZ8. Asterisks indicate millions per cauda epididymis (D, E, and F) and percentage sperm motility signiﬁcant differences compared with the controls (*P!0.05 and (G, H, and I) after 60 days of PPT (A, D, and G), DPN (B, E, and H) and PHTPP ***P!0.0001).

Table 1 Terminal body weight and reproductive organ weights of adult rats treated with different doses of PPT, DPN and PHTPP for 60 days. The values for body weight, testis, epididymis, prostate, seminal vesicles and coagulating glands are in grams and the values

for pituitary are in milligrams. Each value represents meanGS.E.M. nZ8

Treatment Terminal body Seminal Coagulating groups weight Testis Epididymis Prostate vesicles glands Pituitary

Control 426.4G8.71 3.965G0.089 1.32G0.02 0.834G0.05 0.529G0.02 0.234G0.02 10.26G0.28 0.05 PPT 417.5G14.97 3.85G0.12 1.265G0.03 0.76G0.02 0.491G0.02 0.237G0.02 12.95G0.48† 0.1 PPT 412.8G8.34 3.886G0.08 1.266G0.03 0.695G0.03 0.502G0.01 0.23G0.01 13.28G0.33* 0.2 PPT 373.8G7.26† 3.746G0.04 1.146G0.02‡ 0.699G0.02 0.494G0.01 0.207G0.01 12.85G0.34† 0.4 PPT 343G8.05‡ 3.481G0.06‡ 1.086G0.02‡ 0.532G0.02† 0.372G0.02‡ 0.137G0.01‡ 12.94G0.44† 0.05 DPN 435.2G3.76 3.807G0.04 1.208G0.02† 0.752G0.02 0.493G0.01 0.226G0.01 12.62G0.36† 0.1 DPN 400G4.3 3.764G0.02 1.226G0.01† 0.811G0.03 0.473G0.01 0.198G0.01 12.4G0.26† 0.2 DPN 392G4.36 3.777G0.08 1.236G0.03 0.782G0.02 0.587G0.02 0.269G0.01 12.58G0.59* 0.4 DPN 401G10.3 3.851G0.03 1.229G0.03 0.85G0.03 0.591G0.01 0.296G0.02 13.09G0.47† 0.05 PHTPP 423.2G8.62 3.997G0.06 1.347G0.02 0.915G0.05 0.541G0.01 0.219G0.01 10.8G0.1 0.1 PHTPP 401.3G5.88 3.805G0.08 1.228G0.02 0.873G0.08 0.484G0.02 0.217G0.02 11.55G0.22 0.4 PHTPP 422.5G11.24 3.664G0.09 1.234G0.02 0.772G0.06 0.462G0.03 0.215G0.02 10.79G0.87 0.8 PHTPP 412.3G12.39 3.916G0.08 1.316G0.02 0.546G0.06* 0.459G0.02 0.21G0.01 11.1G0.73

Signiﬁcantly different from control (*P!0.05, †P!0.01 and ‡P!0.0001).

ABC8 8 8

6 6 6 ** 4 4 4 FSH (ng/ml) FSH (ng/ml) 2 FSH (ng/ml) 2 2

0 0 0 P PN Control Control Control 0.1 PPT 0.2 PPT 0.4 PPT 0.1 D 0.2 DPN 0.4 DPN 0.05 PPT 0.05 DPN 0.1 PHTPP0.4 PHTPP0.8 PHTP 0.05 PHTPP DEF1.5 1.5 1.5

1.0 1.0 1.0

* LH (ng/ml) LH (ng/ml)

0.5 0.5 LH (ng/ml) 0.5

0.0 0.0 0.0

P TPP Control Control Control 0.1 PPT 0.2 PPT 0.4 PPT 0.1 DPN 0.2 DPN 0.4 DPN 0.05 PPT 0.05 DPN 0.1 PHTP 0.4 PH 0.8 PHTPP 0.05 PHTPP GHI 40 20 8 *** * 30 15 6

20 * 10 4

10 5 2 * Prolactin (ng/ml) Prolactin (ng/ml) Prolactin (ng/ml)

0 0 0

Control Control Control 0.1 PPT 0.2 PPT 0.4 PPT 0.1 DPN 0.2 DPN 0.4 DPN 0.05 PPT 0.05 DPN 0.1 PHTPP0.4 PHTPP0.8 PHTPP 0.05 PHTPP

Journal of Endocrinology JKL 15 15 15

10 10 10

* 5 5 5 * * ** Testosterone (ng/ml) Testosterone (ng/ml) Testosterone (ng/ml) 0 0 0

ontrol C Control Control 0.1 PPT 0.2 PPT 0.4 PPT 0.1 DPN 0.2 DPN 0.4 DPN 0.05 PPT 0.05 DPN 0.1 PHTPP0.4 PHTPP0.8 PHTPP 0.05 PHTPP MNO 150 80 80

60 60 100 *

40 40 50 20 20 Estrogen (pg/ml) Estrogen (pg/ml) Estrogen (pg/ml)

0 0 0

T PT ontrol Control C Control 0.1 P 0.2 PPT 0.4 PP 0.1 DPN 0.2 DPN 0.4 DPN 0.05 PPT 0.05 DPN 0.1 PHTPP0.4 PHTPP0.8 PHTPP 0.05 PHTPP

Figure 3 Hormonal proﬁle of serum FSH (A, B, and C), LH (D, E, and F), prolactin treatment. Values are meanGS.E.M. nZ8. Asterisks indicate signiﬁcant (G, H, and I), testosterone (J, K, and L), estrogen (M, N, and O) after 60 days difference over the control (*P!0.05 and **P!0.01). of PPT (A, D, G, J, and M), DPN (B, E, H, K, and N) and PHTPP (C, F, I, L, and O)

Discussion after 30 days of PPT treatment. This indicates that POL could be brought about by the two ERs through different The effects of E can be mediated by the ERs present in the 2 mechanisms. The results of studies investigating the germ cells in a direct autocrine manner or indirectly by effects of E administration on fertility have indicated affecting Sertoli and Leydig cell function (Shaha 2008). On 2 total loss of potency to be the main reason for the the basis of their localisation in the different cell types in infertility observed (Rao & Chinoy 1983, Gill-Sharma the testis, the two ERs can have distinct physiological roles et al. 2001). Hence, the effects of such treatments on the and can mediate different responses. In the present study, pre- and post-implantation embryo loss have not been we have attempted to understand the individual roles of investigated. Administration of tamoxifen, a selective ER the ERs in various aspects contributing towards male modulator (SERM), which can affect both the ERs, to adult fertility. In our study, we report that treatment of adult male rats also causes an increase in preimplantation male rats with ERa- and ERb-selective agonists and ERb antagonist decreases fertility. An increase in PIL and POL (approximately 60%) and post-implantation (approxi- and a consequent decrease in litter size were observed mately 10%) loss with a decrease in litter size and fertility upon treatments with both the agonists. However, upon (Gill-Sharma et al. 1993, Balasinor et al. 2001). Taken ERb antagonist treatment, an increase in only PIL was together these results indicate that although normal observed with no signiﬁcant decrease in litter size, which estrogen signaling through its receptors is important for could be because the increase in PIL was very subtle. PIL maintenance of spermatogenesis and male fertility, over- was the most sensitive fertility parameter since an increase activation, as in the case of agonists treatment, can be in its incidence was observed for all treatments after both detrimental. 30 and 60 days of treatment. This indicates that post- In several studies have interesting hypotheses have meiotic early spermatids were likely to be affected and been proposed to explain the decrease in male fertility

attenuation of E2 signaling though either of the ERs could parameters upon estrogen exposure. For example, lead to PIL. The POL was observed after 60 days of PPT and decreases in steroid receptor expression and sperm nuclear

DPN treatment (Fig. 1B and E), indicating that germ cells condensation, on exposure to E2 and tamoxifen respect- at earlier stages of spermatogenesis, like spermatogonia ively, have been associated with disturbed spermatogen- and spermatocyes, could be affected by these treatments esis and decreased fertility (Aleem et al. 2005, Kaushik et al. (Dixon & Hall 1982). An increase in POL was also observed 2010). Also, aberrant sperm DNA methylation pattern of Journal of Endocrinology

80 *

C D 20 failed spermatids

0 Percentage of tubules showing

DPN Control Bm 0.1 DPN 0.2 DPN 0.4 0.05 DPN

Figure 4 Representative haematoxylin and eosin stained 5 mm testicular sections ‘Bm’ indicates basal membrane, ‘Lu’ indicates lumen. Scale bar represents showing seminiferous tubules at stage IX–XI from control (A), PPT (B), DPN (C), 20 mm. (E) Signiﬁcant increases in the percentages of Stage IX–XI tubules PHTPP (D) treated groups. Failed spermatids were observed upon treatment showing failed spermatid(s) (*P!0.05). Values are meanGS.E.M. nZ5. A full with 0.05 and 0.1 mg/kg per day DPN as indicated by the arrows. colour version of this ﬁgure is available at http://dx.doi.org/10.1530/JOE-15-0045.

imprinted genes have been reported on treatment with observed with E2 and DES administration (Gill-Sharma SERMs, tamoxifen and bisphenol A (BPA) (Pathak et al. et al. 2001; Goyal et al. 2001). The serum testosterone 2009, 2010, Doshi et al. 2013) and have been correlated levels at higher doses of PPT were reduced to 25% of the with the sub-fertility observed after these treatments control values (Fig. 3J); this could be due to the combined (Gill-Sharma et al. 1993, Balasinor et al. 2001, Salian et al, effect of the decrease in serum LH and local effects of ERa 2009). The precise causes contributing to the decrease in on Leydig cell steroidogenesis (Akingbemi et al. 2003). fertility upon administration of the two agonists in our However, regulation by ERb seems to be dose-dependent study are currently being pursued. as only one of the lower doses of 0.1 mg/kg per day DPN In rodents, male sexual behaviour and ability to mate and the highest dose of 0.8 mg/kg per day PHTPP (Fig. 3K

is under the inﬂuence of the testosterone, yet E2 is also and L) have caused a 50% decrease in serum testosterone thought to play an important role (Freeman & Rissman levels. This could be due to the direct effect on Leydig cells 1996, McCarthy & Albrecht 1996). It is evident that there as the serum LH levels are not changed. The levels of

was a decrease in potency in the treatment groups where serum E2 itself were not affected in any of the dosage there was a decrease in serum testosterone levels (Fig. 2A, groups with the exception of the highest dose of 0.4 mg/kg

B, and C). Interestingly, there was a complete lack of per day PPT where there is an increase in the E2 levels (Fig. potency after treatment with the highest concentration of 3M, N, and O). This could be due to changes in aromatase,

0.4 mg/kg per day PPT. Although the serum testosterone since exogenous administration of E2 to adult rats, levels were not signiﬁcantly different at the highest dose increases intratesticular levels of aromatase (Bharti et al.

from those for the other PPT treatment groups, there was 2013) and activation of E2 signaling speciﬁcally through

a signiﬁcant increase in serum E2 levels at this dose. This ERa is known to upregulate aromatase activity in other

ﬁnding indicates that the increase in E2 could have a tissues (Villablanca et al. 2013). Taken together, these

suppressive effect on sexual behaviour in male rats. results indicate that activation of E2 signaling through ERa

Exogenous administration of E2 (Gill-Sharma et al. suppresses the levels of gonadotropins and testosterone, 2001) and exposure to the synthetic estrogen diethyl- thus the observed decrease in fertility could be a stilbestrol (DES) (Goyal et al. 2001) causes a decrease in cumulative effect of HPT axis disruption and local gonadotropin hormones. However, the treatments with steroidogenic defects on spermatogenesis. However, acti-

selective ligands affected the HPT axis differently. Serum vation of E2 signaling via ERb did not affect the FSH and LH levels were signiﬁcantly decreased upon PPT gonadotropin levels thus the decreased fertility could be Journal of Endocrinology treatment, whereas there was no change as a result of due to localised effects on the reproductive tract. treatment with DPN and PHTPP (Fig. 3A, B, C, D, E, and F). Reports of several studies have described a sharp

These results concur with results published in earlier decline in caudal sperm counts and motility on E2 reports indicating that the negative feedback regulation administration (Rao & Chinoy 1983, Gill-Sharma et al.

of gonadotropins by E2 could be mediated through 2001) and exposure to synthetic estrogen DES (Goyal et al. ERa (Lindzey et al. 1998). Interestingly, ERa is also the 2001) and BPA (Salian et al. 2009). Caudal sperm counts predominant ER in the gonadotrope cells in the were affected by all doses of PPT treatment and lower doses

pituitary (Mitchner et al. 1998). E2 is known to be a of DPN treatment (Fig. 2D and E) which could be due to major regulator of prolactin gene expression and release reduced sperm production in the testis, apoptosis of germ

(Dannies 1985), and E2 treatment increases serum prolac- cells or retention of spermatids in the seminiferous

tin levels (Gill-Sharma et al. 2001). An increase in serum epithelium, all of which are typical effects of E2 exposure prolactin was observed with both the agonists and a (D’Souza et al. 2005). The exact reasons for the low corresponding decrease was found with the antagonist counts are currently being investigated. In the present

PHTPP (Fig 3G, H, and I), indicating that the effects of E2 study, sperm motility was affected by lower doses of DPN on prolactin levels are brought about by both the ERs in a (Fig. 2H) which could be due to testicular defects and/or ligand-dependent manner. Results of recent in vitro studies defects in maturation of the sperm in the epididymis. also support this observation that prolactin gene Although sperm motility was not affected by PPT under expression can be promoted by both the ERs through the experimental conditions examined, the involvement their respective agonists (Chen et al. 2009b). of ERa cannot be ruled out. Steroidogenic enzymes responsible for production of The terminal body weight were unaltered by DPN and

androgens are known to be regulated by E2 (Sakaue et al. PHTPP treatment and decreased only with PPT. E2 2002) and a decrease in testosterone levels is commonly administration has been known to affect weight gain in

males (Robaire et al. 1987), and results from some recent attenuate the age-related decline in spermatogenesis

studies have indicated that the anorexigenic effect of E2 in (Hamden et al. 2008, Clarke & Pearl 2014). On the other

females is modulated specifically through ERa and not ERb hand, overexposure to E2, synthetic estrogens, like DES, (Santollo & Eckel 2009, Santollo et al. 2010). The weights and SERMs, like tamoxifen and BPA, cause deleterious of most of the reproductive organs were more or less effects on the male reproductive tract and fertility. Thus, a unaffected by the treatments except in the 0.4 mg/kg per balance of estrogen action is essential and either deficit or day PPT dose group where the weights of prostate, seminal excess would be detrimental to male fertility. vesicles and coagulating glands are significantly reduced

(Table 1). This could be due to both increased E2 and decreased testosterone levels. There was a decrease in the Conclusion epididymal weights with the higher doses of PPT and lower

doses of DPN which could be due to the decreased sperm The results of this study indicate that overactivation of E2 counts observed after these treatments. Interestingly, a signaling through both the ERs can affect male fertility but signiﬁcant decrease in the weight of prostate gland was presumably through different mechanisms. The effects observed upon treatment with 0.8 mg/kg per day PHTPP. observed after administration of an ERa agonist, are Since the prostate gland shows the highest expression of mediated mainly through HPT axis, whereas treatment ERb in the male reproductive tract in rats (Kuiper et al. with an ERb agonist mainly affects the male reproductive 1997), disruption of its function by the antagonist tract directly. The most interesting ﬁnding from the

treatment could lead to the observed reduction in the present study is that overstimulation of E2 signaling weight of the prostate gland. There was also a dose- through ERb alone, as in the case of 0.05 DPN treatment, dependent increase in weights of pituitary upon treatment is sufﬁcient to cause a decrease in most of the fertility with both the agonists. Similar effects on pituitary have parameters like litter size, sperm counts and motility,

also been observed with exogenous E2 administration and without affecting the levels of any other hormones, or this supports the well-established observation that mito- sexual behaviour. The results of this study indicate that

genic activity of E2 causes a signiﬁcant increase in treatments with ERa and b selective agonists, which cause lactotrophs and, due to their prevalence, a commensurate a decrease in fertility, can be used as models to further increase in pituitary weight (Dannies 1985). delineate the individual roles of the two receptor subtypes Although no visible effect on testicular morphology and the molecular mechanisms involved therein. Journal of Endocrinology was observed at the light microscopy level as a result of PPT treatment, changes in different testicular cell populations cannot be ruled out. An increase in the number of stage Declaration of interest IX–XI tubules showing failed spermatids towards the basal The authors declare that there is no conﬂict of interest that could be perceived as prejudicing the impartiality of the research reported. membrane was observed after treatment with 0.05 and 0.1 mg/kg per day DPN (Fig. 4E). This could be one of the reasons for the observed low sperm counts upon DPN Funding treatment. Surprisingly, this effect was not seen with PPT This study (RA/179/08-2014) has been funded by the National Institute for treatment, despite the reduced testosterone and FSH levels. Research in Reproductive Health (NIRRH) core budget.

This indicates that E2 through ERb, along with testosterone, plays an important role in the process of spermiation Acknowledgements and activation of E2 signaling through ERb alone can cause The authors gratefully acknowledge Indian Council of Medical Research retention of mature spermatids. The results of parallel (ICMR) for providing ﬁnancial support and fellowship to Ms K Dumasia. investigations in our lab have also indicated that cyto- The authors appreciate the technical expertise and assistance of skeletal genes regulated by ERb are involved in spermiation Mr S M Mandavkar in animal experiments, dissections and histology work. The authors are grateful for the technical assistance provided by and their downregulation leads to failure of spermiation Ms G Suryavanshi, Ms S Deshpande and Mr D Shelar during the course of (Kumar et al. 2015). this work.

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Received in ﬁnal form 13 March 2015 Journal of Endocrinology Accepted 2 April 2015 Accepted Preprint published online 13 April 2015