Testicular Hypoplasia with Normal Fertility in Neudesin-Knockout Mice

Abstract

Neudesin is a secretory protein involved in the brain development during embryonic period and diet-induced development of adipose tissue. Although neudesin is also expressed in the testis, its physiological functions in the testis have not been documented. Therefore, we examined neudesin-encoding neuron-derived neurotrophic factor (Nenf) gene-knockout (Neudesin-KO) mice to clarify the functions of neudesin in the testis. The testicular size of the Neudesin-KO mice was significantly smaller than that of wild-type (WT) mice. However, histological analyses did not reveal any abnormalities in the testis, caput epididymis, and cauda epididymis. Sperm number in the cauda epididymis was comparable between WT and KO mice. Neudesin-KO male mice produced vaginal plugs on paired WT female mice, with a frequency similar to that in WT male mice. A similar number of embryos were developed in the females copulated with WT and Neudesin-KO males. Molecular analysis indicated that the ion transporters Slc19a1 and Kcnk3 were more expressed in the testis of Neudesin-KO mice than in the testis of WT mice, suggesting that the transport of ions and some nutrients in the testis has some abnormalities. Testicular size decreased on postnatal day 6, but not on the day of birth, indicating that neudesin is involved in the postnatal, but not embryonic, development of testis. These results indicate a novel role of neudesin in the development of testis.

INTRODUCTION

Neudesin, also known as neuron-derived neurotrophic factor (NENF), is a secretory protein expressed in various organs. It was originally found to be expressed in the embryonic brain and spinal cord.¹⁾ In vitro studies have indicated that in the nervous system, neudesin has neurotrophic and neuroprotective functions.^2,3) The functions of neudesin at the animal level have been investigated by generating Neudesin-knockout (KO) mice. These animals appeared healthy and fertile without any obvious morphological abnormalities.^4,5) Behavioral experiments indicated that Neudesin-KO mice showed an anxious-like behavior with reduced dopaminergic input and dendritic arborization of dentate gyrus neurons, which is consistent with the expression of neudesin in the embryonic and adult brains and the results of in vitro studies.⁴⁾ In contrast, neudesin is also highly expressed in adipose tissues, and white and brown adipose tissues are less developed in Neudesin-KO mice fed with a high-fat diet.^6,7) Furthermore, neudesin is also expressed in other organs, including the lungs, kidneys, skeletal muscles, stomach, and testes, in mice.¹⁾ However, the physiological functions of neudesin in these organs have not yet been explored.

In this study, we examined the testicular phenotypes of Neudesin-KO mice. The results showed that the Neudesin-KO mice had smaller testes, with fertility comparable to that in their wild-type (WT) counterparts, indicating that neudesin plays a significant role in determining testicular size without affecting spermatogenesis.

MATERIALS AND METHODS

Animals

The Neudesin-KO mouse line was generated in Kyoto University.⁵⁾ The Neudesin-KO and control WT mice on the C57BL/6n genetic background were maintained at the animal facility of Kobe Pharmaceutical University. They were individually housed in separate cages for at least 1 week before the experiments. All procedures of the animal experiments in this study were conducted following the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan. The protocols were approved by the Kobe Pharmaceutical University Committee for Animal Care and Use.

Mating Experiment

Eight- to ten-week-old WT and Neudesin-KO males were paired with 8-week-old WT virgin females. The presence of a vaginal plug was monitored every morning for the following 7 d. Plugged females were separated from the males, and another virgin female was paired with each male. The plugged day was designated as gestation day (GD) 0. They were deeply anesthetized with isoflurane and euthanized by cervical dislocation to examine the number of embryos at GD15–17.

Morphological and Histological Analyses

Adult and newborn mice were deeply anesthetized with isoflurane and euthanized by cervical dislocation. Then, the tissues were dissected and weighed. The testes of newborn pups were too small to be weighed; therefore, their volumes were calculated using an ellipsoid volume formula (1/2 × major axis × minor axis²) from the overview pictures. Statistical significance of tissue weight and testicular volume between WT and Neudesin-KO mice was evaluated using Students’ t-test.

Epididymal sperm were dispersed in phosphate-buffered saline (PBS), placed on a hemocytometer, and counted under an Axio Scope A1 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).

For the histological analyses, the mice were cardially perfused with 4% paraformaldehyde in PBS under an isoflurane anesthetic, according to our previous manuscript.⁸⁾ The testes of these animals were post-fixed in 4% paraformaldehyde in PBS at 4 °C. For the Giemsa staining, the testes were cryoprotected in 30% sucrose in PBS at 4 °C overnight. Then, 30-µm tissue sections were prepared in a cryostat (SLEE medical GmbH, Mainz, Germany). The sections were briefly washed in water and immersed in a Giemsa staining solution (Nacalai Tesque, Inc., Kyoto, Japan) at room temperature for 60 min. Then, they were differentiated in 0.5% acetic acid solution, dehydrated by sequentially immersing them in isopropanol twice and xylene five times, and mounted with Entellan^® New (Merck & Co., Inc., Rahway, NJ, U.S.A.). The preparation of paraffin-embedded sections and hematoxylin–eosin (H&E) staining were performed by Genostaff, Co., Ltd. (Tokyo, Japan). Images were captured using an Axio Scope A1 microscope and processed in GNU Image Manipulation Program (GIMP), an open resource software for manipulating images.

RNA Purification and Quantitative (q)RT-PCR

RNA extraction and qRT-PCR analysis were performed as described in a previous manuscript with minor modifications.⁹⁾ Briefly, the mice were deeply anesthetized with isoflurane and euthanized by cervical dislocation. The testes of these animals were dissected, minced using scissors, and frozen in Sepasol-RNA I super G solution (Nacalai Tesque). The tissues were homogenized, and total RNA was purified according to the manufacturer’s instructions. The concentrations of purified RNA were determined using a NanoDrop 1000 spectrophotometer (Thermo-Fisher Scientific, Inc., Wilmington, DE, U.S.A.). cDNA was synthesized using ReverTra Ace reagent (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions. The expression of target genes was determined using a CFX Connect real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.). PCR amplification was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories) with the following PCR parameters: 1 min of initial DNA polymerase activation and DNA denaturation at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 s, and primer annealing at 60 °C for 30 s. The melting curves of the PCR products were analyzed from 65 to 95 °C. Differences in gene expression, expressed as fold changes, were calculated using the ∆∆Ct method, where Rplp2 was used as the reference gene for normalizing the expression, using Excel (Microsoft, Co., Redmond, WA, U.S.A.).¹⁰⁾ The data were presented as the fold changes to the average values of WT. Statistical significance was evaluated using Students’ t-test. The primers were synthesized by Thermo-Fisher Scientific, and their sequences are listed in Supplementary Table 1.

Table 1. Tissue Weights of Neudesin-KO Mice at 4-Week-Old

Tissue	WT (% of b.w.)	KO (% of b.w.)	p-Value
Brain	2.729 ± 0.16	2.663 ± 0.33	0.66
Thymus	0.420 ± 0.080	0.465 ± 0.043	0.25
Lung	0.687 ± 0.028	0.739 ± 0.072	0.14
Kidney	0.683 ± 0.039	0.717 ± 0.049	0.21
Liver	5.944 ± 0.315	5.001 ± 0.914	0.053
Epididymal fat	0.523 ± 0.085	0.661 ± 0.183	0.13
Testis	0.333 ± 0.030	0.257 ± 0.018	2.9 × 10−4 ***

b.w.: body weight. *** p < 0.001.

RESULTS

Reduced Testicular Size in Neudesin-KO Mice

To reveal the functional roles of neudesin in the testis, we examined the size and morphological features of the testes of adult Neudesin-KO mice. The average body weight was comparable between WT and Neudesin-KO male mice (WT: 16.5 ± 1.15 g vs. KO: 16.8 ± 1.99 g, at 4-week-old; WT: 27.1 ± 2.92 g vs. KO: 28.3 ± 2.82 g, at 8-week-old). Together with the previous report of body weight on embryonic day 18.5,⁷⁾ neudesin is dispensable for the fetal and postnatal increase of body weight. The overall view of the testes indicated that the testes of Neudesin-KO mice were apparently smaller than those of WT mice (Fig. 1). The examination of tissue weight revealed a significant reduction in the weight of the testes of Neudesin-KO mice, although the weights of their brain, thymus, heart, spleen, kidney, and liver were comparable with those of WT mice. The lung and epididymal fat of Neudesin-KO mice were rather heavier than those of WT mice at 4-week-old, although the differences were not statistically significant (Tables 1, 2).

Fig. 1. Smaller Testicular Size in Neudesin-KO Mice

Whole view of the testis of 4-week-old (A) and 8-week-old (B) male mice. Two testes from different male mice for each genotype are presented. Scale bars, 5 mm.

Table 2. Tissue Weights of Neudesin-KO Mice at 8-Week-Old

Tissue	WT (% of b.w.)	KO (% of b.w.)	p-Value
Heart	0.465 ± 0.048	0.476 ± 0.029	0.69
Spleen	0.283 ± 0.071	0.271 ± 0.030	0.77
Kidney	0.670 ± 0.055	0.691 ± 0.069	0.65
Liver	4.208 ± 0.593	4.608 ± 0.241	0.26
Testis	0.375 ± 0.014	0.306 ± 0.046	0.029*

b.w.: body weight. * p < 0.05.

Giemsa staining of the tissue sections of the testes of WT and Neudesin-KO mice indicated that the seminiferous tubules of Neudesin-KO mice had normal tubule structure and developing spermatocytes (Figs. 2A, D). Spermatogenic cycle in the seminiferous tubules was observed in both WT and Neudesin-KO mice, and the histological features at each stage of spermatogenesis were not obviously affected in the Neudesin-KO mice (Figs. 2G–N). The caput epididymis and cauda epididymis of Neudesin-KO mice were also comparable to those of WT mice (Figs. 2B, C, E, F). The sperm in the caput epididymis had normal head and tail shapes (Figs. 2B´, E´). Sperm numbers in the cauda epididymis were not different according to genotype (1.02 × 10⁶ ± 0.100 × 10⁶ sperm per cauda epididymis in WT mice [n = 6] vs. 0.920 × 10⁶ ± 0.161 × 10⁶ sperm per cauda epididymis in Neudesin-KO mice [n = 5]).

Fig. 2. Normal Histological Features of the Testis and Epididymis of Neudesin-KO Mice

A–F: The testis (A, D), caput epididymis (B, E), and cauda epididymis (C, F) sections of 8-week-old WT (A–C) and Neudesin-KO (D–F) mice were stained using the Giemsa method. Yellow square regions in panels B and E were magnified in B′ and E′, respectively. G–N: The seminiferous epithelium of 8-week-old WT (G–J) and Neudesin-KO (K–N) mice stained by H&E staining. The images at stage I–III (G, K), V–VI (H, L), VIII (I, M), and X–XII (J, N) are presented. Scale bars, 100 µm.

Although Neudesin-KO mice have been implicated to be fertile in previous manuscripts, we quantitatively examined their fertility. When WT and Neudesin-KO male mice were paired with WT females, they produced vaginal plugs with similar frequency (Fig. 3). Some WT and Neudesin-KO males did not produce vaginal plugs during the whole experimental period (WT#2 and Neudesin-KO#2, #4). The reason of this failure of pairing is unknown at this stage. The plugged females were pregnant and produced a similar number of embryos at the late gestation stages (Fig. 4). Thus, the Neudesin-KO male mice had normal fertility.

Fig. 3. Detection of Vaginal Plugs in Females Paired with WT or Neudesin-KO Males

Seven WT and eight Neudesin-KO males were each paired with one WT female at 8-week-old. The circles indicate the vaginal plug-positive females.

Fig. 4. Number of Embryos in Females Copulated with WT or Neudesin-KO Males

The vaginal plug-positive females were maintained up to GD15-17. The number of embryos were examined as described in the Materials and Methods section. Error bars indicate standard deviation.

Excess mRNA Expression of Ion Transporters in the Testes of Neudesin-KO Mice

To determine abnormalities in the testes of Neudesin-KO mice at the molecular level, we examined mRNA expressions in the testes. Our preliminary transcriptome analysis of the brain of Neudesin-KO mice indicated that some mRNAs related to membrane ion transport are affected by the lack of the Neudesin gene (Kondo et al., unpublished data). Therefore, we examined the expression of several ion transporters in the testes of WT and Neudesin-KO mice using the qRT-PCR method (Table 3). Only traces of the Neudesin mRNA, as a reference, were observed in the testes of Neudesin-KO mice. Among the mRNAs examined, Slc19a1 and Kcnk3 were significantly upregulated by the lack of the Neudesin gene. In contrast, Slc28a3 tended to be downregulated in the testis of Neudesin-KO mice, although no statistical significance was detected.

Table 3. Relative Expression of mRNAs Encoding Ion Transporters in WT and Neudesin-KO Testis at 8-Week-Old

Gene	WT	Neudesin-KO	p-Value
Neudesin	1.00 ± 0.104	0.000156 ± 0.000312	1.26 × 10−6 ***
Slc3a1	1.00 ± 0.214	1.35 ± 0.291	0.102
Slc4a5	1.00 ± 0.209	1.19 ± 0.121	0.172
Slc4a11	1.00 ± 0.250	1.36 ± 0.275	0.103
Slc6a11	1.00 ± 0.164	0.87 ± 0.153	0.291
Slc16a11	1.00 ± 0.460	1.49 ± 0.138	0.0858
Slc19a1	1.00 ± 0.381	1.90 ± 0.341	0.0123 *
Slc25a29	1.00 ± 0.598	1.83 ± 0.641	0.107
Slc28a3	1.00 ± 0.191	0.69 ± 0.178	0.0565
Kcnk3	1.00 ± 0.372	2.47 ± 0.517	0.00364 **
Kcnq5	1.00 ± 0.363	1.29 ± 0.182	0.204

* p < 0.05, ** p < 0.01, *** p < 0.001.

Onset of Size Reduction of the Testes of Neudesin-KO Neonates

To determine the developmental stage at which the abnormality of testicular size is first observed, we analyzed postnatal day 0 (PND0) and 6 (PND6) neonates. The testicular size was comparable between WT and Neudesin-KO PND0 neonates (Fig. 5A and Table 4). In contrast, the testes of PND6 mice were significantly smaller than those of WT PND6 mice (Fig. 5B, Table 4). The reduction of testicular volume is maintained by adult (Table 4). Thus, the embryonic development of the testis is independent of the Neudesin gene; however, neudesin is required for the normal development of the testis at neonatal stages.

Fig. 5. Developmental Hypoplasia in the Testes of Neudesin-KO Mice

The testes of PND0 (A) and PND6 (B) WT and Neudesin-KO neonates. The overview of the testis, with the epididymis, is shown. Scale bars, 1 mm.

Table 4. Testicular Volume during Development of Neudesin-KO Mice

Stage	WT (mm3)	KO (mm3)	p-Value
PND0	0.741 ± 0.124	0.773 ± 0.064	0.66
PND6	3.705 ± 0.658	2.278 ± 0.445	0.011*
PND14	10.81 ± 1.21	9.00 ± 1.05	0.020*
4-Week-old	60.01 ± 6.15	47.95 ± 5.98	0.0063**

* p < 0.05, ** p < 0.01.

DISCUSSION

In this study, we reported that the testes of Neudesin-KO mice had developmental hypoplasia. The histological and behavioral examinations revealed that Neudesin-KO male mice retained their fertility, indicating that neudesin is dispensable for the functional development of the testis and sperm. The molecular analysis suggested the abnormal expression of some ion channels, which could affect some aspects of testis physiology.

Functions of Neudesin in Testis Development

Neudesin has a neurotrophic effect on neurons, but not on astrocytes, and plays a significant role in the neuronal differentiation of neural progenitor cells.^1,2) These functions of neudesin, examined in cultured cells, were mediated by the activation of the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase A (PKA) pathways, suggesting that neudesin acts through unknown membrane G protein-coupled receptors.⁵⁾ The cAMP–PKA–cAMP response element binding protein (CREB) pathway is important for testicular development.¹¹⁾ Particularly, this pathway is activated by follicle-stimulating hormone (FSH), which plays a central role in testicular development and size determination.^12–15) Thus, neudesin may coordinately regulate testicular development along with FSH through the cAMP signaling pathway.

Alternatively, neudesin may exert its function in testicular development through membrane receptor-independent mechanisms. Neudesin belongs to the membrane-associated progesterone receptor family, which consists of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, and neuferricin. They commonly have a cytochrome b5-like heme/steroid-binding domain (Cyt-b5 domain).¹⁶⁾ The Cyt-b5 domain of PGRMC1 and PGRMC2 has high binding affinity to progesterone.^17–19) Functionally, PGRMC1 has been reported to mediate the anti-apoptotic function of progesterone in granulosa cells.²⁰⁾ Furthermore, PGRMC1 might be involved in estrogen signaling, as it promotes the estrogen-dependent proliferation of breast cancer cells.^21,22) Although whether the Cyt-b5 domain of neudesin has a binding activity to progesterone or any other steroid hormones remains unknown, the studies referred above suggest that neudesin modulates steroid hormone activities.

The hypoplasia of testis in Neudesin-KO mice was observed during first postnatal week, but not on the day of birth, and the hypoplasia is specific to the testis among the tissues examined (Fig. 5, Tables 1, 2). The cause of this tissue-specific hypoplasia is not apparent and should be examined in the following study. Testicular size is largely controlled by testosterone.²³⁾ Testosterone production in the testis is dynamically changed after birth.^24–26) On the other hand, progesterone and estrogen also directly affect the testicular development.²⁷⁾ Thus, it is possible that the change of steroid hormone signalings play a role in the testis-specific developmental hypoplasia in Neudesin-KO mice.

The Functions of Neudesin-Regulating Slc19a1 and Kcnk3 Genes in the Testis

The lack of the Neudesin gene reduced the testicular size but did not affect the histological features and spermatogenic function of the testis. Thus, neudesin is not critically involved in the development of the testis. In contrast, the testis of Neudesin-KO mice showed the increased expression of mRNAs encoding ion transporters Slc19a1 and Kcnk3 mice (Table 3).

SLC19A1 protein, also known as Reduced Folate Carrier 1, is characterized as a high-affinity transporter for reduced folates and antifolates.^28,29) Several studies have indicated the protective actions of folic acid against testicular damage caused by toxic compounds, including bisphenol-A and 3,4-methylenedioxymethamphetamine.^30,31) In addition to folate, SLC19A1 has been implicated to be a major transporter of cyclic dinucleotides, such as cyclic guanosine monophosphate-adenosine monophosphate, which are stimulators of the innate immune response produced in response to infection, cancer, and genomic damage.³²⁾ It will be interesting to know whether neudesin regulates immune response in the testis through the expressional regulation of SLC19A1.

KCNK3, also known as TASK-1, is an acid-sensitive potassium channel. In the embryonic testes, estradiol exposure has been reported to downregulate Kcnk3.³³⁾ Furthermore, in decidual cells, the expression of Kcnk3 is regulated by estrogen and progesterone.³⁴⁾ Thus, Kcnk3 expression is regulated by steroid hormone activities, which could be modulated by neudesin. Kcnk3 mRNA is expressed in Leydig cells, although its function in the testis and steroidogenesis is still unknown.³⁵⁾ During the embryonic development of the testis, fetal Leydig cells produce activin A, which regulates Sertoli cell proliferation and testis cord expansion. Fetal Leydig cells are also involved in testosterone production during postnatal development.³⁶⁾ Although our histological analyses of Neudesin-KO mice did not detect obvious changes in Sertoli cells (Fig. 2, data not shown), it is possible that detailed functions of Sertoli cells are affected in the Neudesin-KO mice, which should be clarified in the following studies.

Acknowledgments

The authors thank Dr. Hirofumi Hohjoh for his efforts in maintaining the environment of the animal facility. This work was supported by the Kobe Pharmaceutical University President’s Discretionary Expenses.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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