1

Original Article

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Spike protein of SARS-CoV-2 suppresses gonadotrophin secretion from bovine anterior

3

pituitaries

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5

Dimas Arya Abdillah1, Onalenna Kereilwe1, Raihana Nasrin Ferdousy1, Risa Saito1 and Hiroya

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Kadokawa1*

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8

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Japan

Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi-shi, Yamaguchi-ken 1677-1,

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Key words: ACE2, anterior pituitary gland, Covid-19, gonadotroph, S1, S2

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Running head: Spike protein and gonadotropin secretion

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14*Corresponding author: Prof. Hiroya Kadokawa, Faculty of Veterinary Medicine, Yamaguchi

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University, Yamaguchi-shi, Yamaguchi-ken 1677-1, Japan.

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Tel.: 083 9335825. E-mail: hiroya@yamaguchi-u.ac.jp

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Abstract

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Coronavirus disease (COVID-19), the ongoing global pandemic, is caused by the severe acute20

respiratory syndrome coronavirus-2 (SARS-CoV-2). Recent evidence shows that the virus utilizes

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angiotensin-converting enzyme 2 (ACE2) as a spike protein receptor for entry into target host cells.

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The bovine ACE2 contains key residues for binding to the spike protein receptor-binding domain. This

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study evaluated the hypothesis that bovine gonadotroph expresses ACE2, and spike protein suppresses

24

luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion from cultured bovine

25

anterior pituitary (AP) cells. ACE2 mRNA expression and ACE2 protein expression were detected in26

the bovine AP cells using reverse transcription PCR and western blot analysis. Immunofluorescence27

microscopy analysis with the anti-ACE2 antibody revealed the co-localization of ACE2 and28

gonadotropin-releasing hormone (GnRH) receptor on the gonadotroph plasma membrane.

29

Approximately 90% of GnRH receptor-positive cells expressed ACE2, and approximately 46% of30

ACE2-positive cells expressed the GnRH receptor. We cultured bovine AP cells for 3.5 days and31

treated them with increasing concentrations (0, 0.07, 0.7, or 7 pM) of recombinant spike protein

32

having both S1 and S2 regions. The spike protein (0.07–7 pM) suppressed both basal and33

GnRH-induced LH secretion (P < 0.05). Spike protein (0.7–7 pM) suppressed GnRH-induced (P <340.05), but not basal FSH secretion. In contrast, pre-treatment with ERK 1/2/5 inhibitor (U0126) partially35

restored the GnRH-induced LH and FSH secretion from the spike protein suppression. Collectively, the36results indicate that gonadotrophs express ACE2, a receptor for coronavirus 2 spike protein, which in37

turn suppresses LH and FSH secretion from AP cells.

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Key words: ACE2, anterior pituitary gland, COVID-19, gonadotroph, S1, S2

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41
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Introduction

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The ongoing global coronavirus disease (COVID-19) pandemic has been caused by the spread of44

severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Recent evidence shows that the virus

45

utilizes angiotensin-converting enzyme 2 (ACE2) as a spike protein receptor to enter target host cells

46[1]; however, the primary role of ACE2 is to convert angiotensin II to angiotensin-(1–7). Cells

47

expressing ACE2 may thus be a target for the virus. ACE2 is highly expressed in the human testes,48

ovaries, and other reproductive organs [2]. Indeed, the virus may be an important cause of infertility in49

men because of its deleterious effects on semen quality and quantity through unclarified mechanisms,50

as reviewed by Agolli et al. [3] and Moshrefi et al. [4]. However, it remains unclear whether the virus

51

also causes female infertility, especially in domestic animals.

52

The anterior pituitary (AP) gland lies outside the blood-brain barrier [5], and may be affected by53

the virus [6]. Gonadotrophs are important cells located in the AP glands, and secrete luteinizing

54

hormone (LH) and follicle-stimulating hormone (FSH), which regulate the testes, ovaries, and, via

55

gonadal steroids, other reproductive organs in animals [7]. However, to the best of our knowledge, the56effect of the virus on gonadotrophs remains unclear. Gu et al. [8] reported that human gonadotrophs

57

express ACE2. A recent study clarified that the spike protein of SARS-CoV-2 directly activates the58

cytoplasmic extracellular signal-regulated kinase (ERK) pathway downstream of ACE2 in human59

platelets [9]. The ERK pathway is also the cytoplasmic pathway downstream of the membrane

60

estradiol receptor (GPR30), that suppresses LH secretion in bovine gonadotrophs [10]. However, the61mechanism by which spike protein-activated ACE2 affects LH and FSH secretion by gonadotrophs in62

all species remains unclear.

63

Cattle are important domestic animals for food supply worldwide; thus, infertility in cattle is an

64

important issue. Unlike human AP glands, bovine AP glands can be obtained for primary culture as

65

they can be collected from slaughterhouses. Indeed, using bovine gonadotrophs, we discovered new66

receptors that control LH and FSH secretion, colocalizing with gonadotropin-releasing hormone

67(GnRH) receptor (GnRHR) in the lipid rafts of bovine gonadotrophs [7, 11, 12, 13]. Bovine ACE2

68

contains most of the key residues for the receptor-binding domain of SARS-CoV-2 [14] (details are

69

shown in Supplementary Fig.1), and SARS-CoV-2 replicates in bovine respiratory tissues [15].

70

Therefore, we tested the hypothesis that gonadotrophs express the spike protein receptor ACE2,

71

colocalizing with GnRHR on the bovine gonadotroph cell surface, and that the recombinant spike

72

protein suppresses LH and FSH secretion from cultured bovine AP cells. It is also important to clarify73

the cytoplasmic signaling pathway downstream of ACE2. Therefore, we used an inhibitor to evaluate74

the contribution of the ERK pathway to the effect of spike protein on gonadotropin secretion from the75

bovine AP.

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77

Materials and Methods

78

Anterior pituitary sample collection

79

All experiments were performed in accordance with the Guiding Principles for the Care and Use

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of Animals in the Field of Physiological Sciences (Physiological Society of Japan). All experiments

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involving animals were approved by the Animal Ethics Committee of Yamaguchi University (approval

82

number 301).

83

We obtained AP tissue from post-pubertal (26 months of age) Japanese Black heifers at a local

84

abattoir, using a previously described method [16]. All heifers were in the luteal phase, as determined85

by macroscopic examination of the ovaries and uterus [11]; the AP gland exhibits the highest LH, FSH,

86

and GnRH receptor levels in this phase [17].

87

The AP samples for RNA or protein extraction (n = 5) were stored at −80°C. The AP samples for88

immunohistochemistry (n = 5) were fixed with 4% paraformaldehyde at 4°C for 16 h. The AP samples

89

to be used for cell culture followed by immunocytochemical analysis (n = 5), and those that were to be90

used for cell culture to evaluate the effect of spike on LH and FSH secretion (n =6) were stored in91

ice-cold 25 mM HEPES buffer (pH 7.2) containing 10 mM glucose and transported to the laboratory92

on ice.

93
94

RT–PCR, sequencing of amplified products, and homology search in gene databases

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Total RNA was extracted using RNAzol RT isolation reagent (Molecular Research Centre,

96

Inc., Cincinnati, OH, USA) and treated with deoxyribonuclease. The concentration and purity of each97

RNA sample was evaluated using spectrophotometry (acceptable range, 1.8–2.1) and electrophoresis

98(28S:18S ratios were 2:1). Complementary DNA was synthesized using the Verso cDNA Synthesis Kit

99(Thermo Fisher Scientific, Waltham, MA, USA).

100

We used previously reported RT-PCR methods [11] to detect the mRNA levels of ACE2 (NCBI

101

reference sequence, NM_001024502). The expected amplicon size of ACE2 was 470 bp (orange

102

highlighted region in Supplementary Fig.1； nucleotides 1297-1766; forward primer in 9th exon:

1035′-CCGCAGCCACACCTCACTAT-

1045′-GGTCCAGGGTTCTGATTTTCC-3′). PCR was performed using 20 ng of cDNA and polymerase

105(Tks Gflex DNA Polymerase, Takara Bio Inc., Shiga, Japan) under the following thermocycling106

conditions: 94°C for 1 min for pre-denaturation, followed by 35 cycles of 98°C for 10 s, 60°C for 15 s,

107

and 68°C for 30 s. The PCR products were separated on 1.5% agarose gel using electrophoresis with a108

molecular marker (Nippon Gene, Tokyo, Japan), stained with Gelstar (Lonza, Allendale, NJ, USA),109

and observed using a charge-coupled device (CCD) imaging system (GelDoc; Bio-Rad, Hercules, CA,

110USA). The PCR products were purified using a NucleoSpin Extract II kit (Takara Bio Inc.) and then111

sequenced using PCR primers and the Dye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher

112

Scientific). The sequences obtained were used as query terms to search the homologous sequences

113

using the basic nucleotide local alignment search tool (BLAST) (available on the NCBI website).

3′;

reverseprimer

in

13th

exon:

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115

Antibodies used in this study116

We used a specific anti-ACE2 rabbit polyclonal antibody (HPA000288; Sigma-Aldrich, St. Louis,

117

MO, USA). The antigen sequence that produces the antibody has 86% homology with the118

corresponding region of bovine ACE2, as shown by the green highlighted region in Supplementary

119

Fig.1. In particular, ACE2 has a single transmembrane region [18], as indicated by the blue highlighted

120

region in Supplementary Fig.1. There is 91% homology between the extracellular regions of bovine

121

and human ACE2 (the NCBI reference sequences of bovine and human ACE2 are NP_001019673.2

122

and BAB40370, respectively).

123

We also used a guinea pig polyclonal antibody that recognizes the N-terminal extracellular domain

124of the bovine GnRH receptor (anti-GnRHR). The specificity of the anti-GnRHR antibody has been

125

verified previously [16]. We used a mouse monoclonal anti-LH antibody (clone 518-B7) [19] and a126

mouse monoclonal anti-FSH antibody (clone A3C12) [20] for immunohistochemical analysis of AP

127

tissue and cultured AP cells. These antibodies do not cross-react with other pituitary hormones [20,

128

21].

129
130
131

Western blot analysis for ACE2
We extracted proteins from AP tissue and performed western blotting using a previously132

described method [16]. Briefly, total proteins were extracted from frozen stock AP tissue using a tissue

133

protein extraction reagent containing Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). The

134

extracted protein sample was boiled with Sample Buffer Solution containing Reducing Reagent (6x)135

for SDS-PAGE (09499-14; Nacalai Tesque, Kyoto, Japan) for 3 min at 100°C. The protein samples

136(8,000 ng of total protein) were loaded onto a polyacrylamide gel (Any KD Criterion TGX gel,137

Bio-Rad) along with the whole-cell lysate of human liver-derived HepG2 cells (sc-2227, Santa Cruz,

138

Heidelberg, Germany) as positive controls [22], and a molecular weight marker (Precision Plus Protein

139

All Blue Standards; Bio-Rad). The proteins were resolved by sodium dodecyl sulfate-polyacrylamide

140

gel electrophoresis at 100 V for 90 min. The proteins were transferred onto polyvinylidene fluoride

141(PVDF) membranes (Trans-blot turbo PVDF, Bio-Rad) with electroblotting at 2.5 A, 25 V, for 7 min

142

using a Trans-blot Turbo system (Bio-Rad).

143
144

A Can Get Signal kit (Toyobo Co. Ltd., Osaka, Japan) was used to block the membrane (1 h
at 25°C), primary antibody reaction (1 h at 25°C) with the anti-ACE2 rabbit antibody (1:400,000

145

dilution with immunoreaction enhancer solution), and secondary antibody reaction (1 h at 25°C) with146

a goat anti-rabbit IgG horseradish peroxidase-conjugated antibody (Bethyl Laboratories, Inc.,

147

Montgomery, TX, USA; 1:400,000 dilution with immunoreaction enhancer solution). The protein

148

bands were visualized using an ECL-Prime chemiluminescence kit (GE Healthcare, Amersham, UK)149

and a CCD imaging system (LAS-3000 Mini; Fujifilm, Tokyo, Japan). To verify the specificity of the150

signals, we included several negative controls in which the primary antibodies were omitted, or normal

151

rabbit IgG (Wako Pure Chemicals, Osaka, Japan) antibodies were used instead of the specific primary152

antibodies. Signal specificity was also confirmed using negative controls in which the primary153

antibodies were pre-absorbed with 4 nM of the antigen peptide (PrEST Antigen ACE2, APREST74018,

154

Sigma-Aldrich).

155

The antibodies were removed from the PVDF membrane using a stripping solution (Nacalai156

Tesque) before blocking and subsequent immunoblotting with an anti-β-actin mouse monoclonal

157

antibody (1:400,000 dilution; Sigma-Aldrich).

158
159

Fluorescence immunohistochemistry and confocal microscopy

160

We followed our previously reported method [16] for the immunofluorescence analysis of AP

161

tissue (n = 5) after storage in 4% paraformaldehyde PBS at 4°C for 16 h, and 30% sucrose PBS until162

the blocks were infiltrated with sucrose. The blocks were frozen in an embedding medium (Tissue-Tek

163

OCT compound Sakura Finetechnical Co. Ltd., Tokyo, Japan) and maintained at -80°C. Briefly,

164

15-µm sections were prepared using a cryostat, mounted on slides for treatment with 0.3 % Triton

165

X-100 in PBS for 15 min, and blocked with 10% normal goat serum in PBS for 1 h. Incubation with a166

cocktail of primary antibodies (anti-ACE2 rabbit antibody, anti-GnRHR guinea pig antibody, and167

either anti-LH or anti-FSH mouse antibody [all diluted 1:1,000]) for 12 h at 4°C was followed by168

incubation with secondary antibodies (Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 546 goat169

anti-mouse IgG, and Alexa Fluor 647 goat anti-guinea pig IgG [all from Thermo Fisher Scientific and170

diluted as 1 µg/mL]) for 2 h at room temperature, and counterstaining with 1 µg/mL of 4,171

6-diamino-2-phenylindole (DAPI; Wako Pure Chemicals).

172

The stained sections were observed using a confocal microscope (LSM710; Carl Zeiss, Göttingen,173

Germany) equipped with diode (405 nm), argon (488 nm), HeNe (533 nm), and HeNe (633 nm) lasers.

174

Images obtained by fluorescence microscopy were scanned with a 40× or 63× oil-immersion objective

175

and recorded using a CCD camera system controlled by ZEN2012 black edition software (Carl Zeiss).

176

To verify the specificity of the signals, we included several negative controls in which the primary177

antiserum had been omitted or pre-absorbed with 4 nM of the antigen peptide, or in which normal

178

rabbit IgG (Wako Pure Chemicals) was used instead of the primary antibody. Ratios of ACE2 positive

179

gonadotrophs were calculated from 12 representative confocal images per AP gland.

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AP cell culture and immunocytochemical analysis of cells

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We followed our previously reported method [16] for the enzymatic preparation of AP cells (n = 6)

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and their culture. Cell viability was confirmed to be greater than 90% by trypan blue exclusion. Total

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cell yield was 19.8 ×106± 0.8 ×106 cells per AP gland. Dispersed cells were then suspended in

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Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific) containing 1 nonessential

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amino acids (Thermo Fisher Scientific), 100 U/mL penicillin, 50 µg/mL streptomycin, 10% horse

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serum (Thermo Fisher Scientific), and 2.5% fetal bovine serum (Thermo Fisher Scientific). The cells

188(2.5 × 105 cells/mL, total = 0.15 mL per lane) were cultured in the culture medium at 37°C in 5% CO2

189

for 82 h, using a microscopy chamber (µ-Slide VI 0.4, Ibidi, Planegg, Germany). Recombinant human190

activin A (final concentration, 10 ng/ml; R&D Systems, Minneapolis, MN, USA) was used to191

stimulate FSH synthesis at 24 h before fixation [11]. FSH secretion from cultured AP cells is weak in

192

ovine and bovine AP cells, and the pre-treatment time and final concentration have already been193

established in previous studies [23].

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The cultured cells were fixed with either 4% paraformaldehyde for 3 min followed by treatment

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with 0.1% Triton X-100 for 1 min (PFA-Triton method), or with CellCover (Anacyte Laboratories UG,

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Kuhreder, Hamburg) for 2 min without Triton X-100 treatment (CellCover method), as described by

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Kadokawa et al. [16]. For the PFA-Triton method, fixed cells were incubated with 0.1 mL of the same198

cocktail of primary antibodies for 2 h at room temperature. Incubation with Triton X-100 allowed

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anti-GnRHR and anti-ACE2 antibodies to bind to target proteins in the cytoplasm and at the cell

200

surface. For the CellCover method, the fixed cells were incubated with guinea pig anti-GnRHR and201

rabbit anti-ACE2 (both 1:1,000) for 2 h at room temperature. As the cells were not treated with Triton

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X-100, the antibodies could only bind to the extracellular domains of the respective receptors in most203

cells. For both the PFA-Triton and CellCover methods, cells were incubated with the204

fluorochrome-conjugated secondary antibody cocktail and DAPI and subjected to confocal microscopy.

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Signal specificity was confirmed using negative controls in which the primary antibody was omitted or

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pre-absorbed at 4 nM with the same antigen peptide. Normal rabbit IgG was used as the primary207

antibody. Eight randomly selected images of cells prepared with the CellCover method were analyzed

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for co-localization using the ZEN 2012 black edition software (Carl Zeiss) to calculate overlap

209

coefficients [24] for Alexa Fluor 488 and Alexa Fluor 647.

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Effects of the recombinant spike protein of SARS-COV-2 on LH and FSH secretion212

AP cells derived from six post-pubertal heifers were plated in 48-well cell culture plates

213(Sumitomo Bakelite, Tokyo, Japan) and incubated at 37°C and 5% CO2 for 82 h. Recombinant human214

activin A (final concentration, 10 ng/mL) was added to the plates 24 h before the test to stimulate FSH

215

synthesis.

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The medium was replaced with 270 µL of DMEM containing 0.1% BSA (IgG-free Protease-free217

culture grade, 032-22364, Wako Pure Chemicals) and 10 ng/mL activin A (base medium) and218

incubated for 2 h to evaluate the effect of spike protein in the absence of GnRH. Treatment was

219

performed by adding 30 µL of base medium alone or 30 µL of base medium with different

220

concentrations of spike protein (final concentrations of 0, 0.07, 0.7, and 7 pM). We used a recombinant

221

spike protein of SARS-CoV-2 containing both the S1 and S2 regions (40589-V08H4; Sino Biological222

US Inc., Wayne, PA, USA). After incubation for 2 h, the medium from each well was collected for223

radioimmunoassay (RIA) analyses of LH and FSH levels.

224

The old medium was replaced with 240 µL of base medium and incubated at 37°C for 2 h to225

evaluate the effect of the spike protein in the presence of GnRH. The cells were pre-treated by adding

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30 µL of base medium containing different concentrations of the recombinant spiked protein (final

227

concentrations of 0, 0.07, 0.7, and 7 pM). The cells were incubated with gentle shaking for 5 min, and228

then treated with 30 µL GnRH (Peptide Institute Inc., Osaka, Japan; final concentration of 1 nM [16])229

dissolved in the base medium for 2 h. As previously reported [16], gonadotropin secretion was

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stimulated by increasing the amounts of GnRH, with a peak at 1 nM of GnRH, and reduced secretion

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at GnRH concentrations higher than 1 nM. Therefore, the final concentration of GnRH used in this232

study was 1 nM in all treatments, except in the controls. After incubation for 2 h, the medium from

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each well was collected for LH and FSH RIAs.

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Effect of ERK pathway inhibitor on the suppression of secretion236

We evaluated the effect of the ERK1/2/5 pathway inhibitor, U0126, on spike-mediated237

suppression of secretion from bovine AP cells. AP cells obtained from a different set of post-pubertal

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Japanese Black heifers (n = 8, in the middle of the luteal phase, 26 months of age), were cultured for239

82 h in the medium described in the previous section. Each experiment was repeated eight times with240

each of the eight AP glands, using four wells per treatment. The wells were washed twice with PBS

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and then incubated with 287 µL of DMEM containing 0.1% BSA and 10 ng/mL activin A for 2 h.

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Cells were pre-treated with 3 µL of DMEM alone or 3 µL of DMEM containing U0126 (final

243

concentration, 1,000 nM; Enzo Biochem, Inc., New York, USA). After 30 min of incubation, either 5244µL of DMEM alone or 5 µL of DMEM containing spike protein (final concentration of 7 pM, which245

showed a significant inhibitory effect on gonadotropin secretion) was added to each culture well. The

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cells were incubated with gentle shaking for 5 min, after which they were incubated for 2 h with 5 µL

247

of DMEM containing GnRH (final concentration, 1 nM) to stimulate gonadotropin secretion. After 2 h

248

of incubation, the medium was collected for RIA. We previously confirmed that pre-treatment with

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1,000 nM U0126 alone had no effect on GnRH-induced gonadotropin secretion, but inhibited the250

ability of estradiol to suppress GnRH-induced gonadotropin secretion from cultured bovine AP cells

251

[10].

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RIAs to measure gonadotropin concentration in culture media

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The concentration of LH was measured in duplicate samples of culture media using

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double-antibody RIA using 125I-labeled bLH and anti-oLH-antiserum (AFP11743B and AFP192279,

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National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and257

Kidney Diseases, Bethesda, CA, USA). The intra- and inter-assay coefficients of variation (CV) were258

3.6% and 6.2%, respectively. The concentration of FSH was measured by double-antibody RIA using

259

bFSH and anti-oFSH antiserum (AFP5318C, AFP5346D, and AFPC5288113, NIDDK). The intra- and260

inter-assay CVs were 4.3% and 7.1%, respectively.

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Statistical analysis

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The statistical significance of differences in LH or FSH concentration was analyzed by one-factor

264

ANOVA followed by post-hoc comparisons using Fisher’s protected least significant difference test in

265

StatView version 5.0, for Windows (SAS Institute, Inc., Cary, NC, USA). The level of significance266

was set at P < 0.05. Data are expressed as the mean ± standard error of the mean (SEM).

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268

Results

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Expression of ACE2 in AP of post-pubertal heifers

270

The expected PCR products were obtained by electrophoresis (Fig. 1A). Homology searching for271

the amplified product sequences revealed that the best match alignment was bovine ACE2

272

(NM_001024502), which was identical (identities were 100% (470/470) with no gaps). No other273

bovine genes were found to have any homology with the obtained sequences of the amplified products,274

suggesting that the sequences of the amplified products were identical to the sequence of bovine

275

ACE2.

276

Western blotting revealed similar bands for AP and HepG2 cells (Fig. 1B). One difference was277

the band size of 42 kDa for AP and 50 kDa for HepG2. Another difference was that a 100-kDa band278

was observed in the AP sample, but not in HepG2. No bands were observed in the negative control279

membranes, where the primary antiserum was pre-absorbed with the antigen peptide.

280
281

Immunofluorescence analysis of ACE2 expression in bovine AP tissue282

ACE2 and GnRHR were colocalized in the majority of LH-positive (Fig. 2A) and FSH-positive283(Fig. 2B) cells in bovine AP tissue. The percentages of single- and double-labeled ACE2- and284

GnRHR-positive cells were determined from 15 representative confocal images per AP gland. In each285

AP gland, there was an average of 54.4 ± 2.4 GnRHR-positive cells, 105.2 ± 1.1 ACE2-positive cells,286

and 48.6 ± 1.7 double-positive cells; further, 89.5 ± 1.6% of GnRHR-positive cells were287

ACE2-positive, and 46.3 ± 1.9% of ACE2-positive cells were GnRHR-positive. No immunostaining288

signals were observed in the negative control tissues, where the primary antiserum was pre-absorbed289with the antigen peptide.

290
291
292
293

ACE2 and GnRHR on the cell surface
Among the AP cells prepared using the PFA-Triton method, we observed ACE2 expression in
LH-positive (Fig. 3A) and FSH-positive cells (Fig. 3B).

294

The AP cells prepared using the cell cover method showed that ACE2 was colocalized on the295

surface of GnRHR-positive cells (Fig. 4). The overlap coefficient of the cultured AP cell surface

296

between ACE2 and GnRHR was 0.73 ± 0.01.

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Effects of spike protein on gonadotropin secretion from cultured AP cells299

Fig. 5 shows the effect of various concentrations of spike protein on LH or FSH secretion from AP

300

cells derived from post-pubertal heifers cultured in the absence (A, C) or presence (B, D) of GnRH. In

301

the absence of GnRH (Fig. 5A), 0.07 pM (P < 0.05), 0.7 pM (P < 0.05), and 7 pM (P < 0.01) of spike

302

protein suppressed LH secretion compared to the controls. Moreover, 0.07 pM (P < 0.05), 0.7 pM (P <303

0.01), and 7 pM (P < 0.01) of spike protein suppressed GnRH-induced LH secretion (Fig. 5B).

304

In the absence of GnRH (Fig. 5C), none of the tested concentrations of spike protein was found to305

suppress FSH secretion compared to the controls. However, 0.7 pM (P < 0.01) and 7 pM (P < 0.01),306

but not 0.07 pM the spike protein suppressed GnRH-induced FSH secretion (Fig. 5D).

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308

Fig. 6 shows that 7 pM of spike protein suppressed GnRH-induced LH secretion, and thatpre-treatment with U0126 partially recovered GnRH-induced LH and FSH secretion.

309
310

Discussion

311

Cultured bovine gonadotroph cells express ACE2, and addition of recombinant spike

312

protein to the culture medium suppressed the secretion of LH and FSH, providing clear evidence313

that the spike protein, containing both S1 (attachment to ACE2) and S2 (fusion with host314

membrane) regions, affects the cytoplasmic ERK pathways [9] that play important roles in315

the control of LH and FSH secretion [10]. ACE2 colocalizes with GnRHR on the lipid rafts of316

gonadotrophs [7], suggesting that the S2 region suppresses LH and FSH secretion by fusing

317

with the lipid rafts [25] to affect heteromer receptors.

318

We found that approximately 90% of gonadotroph cells in bovine AP were ACE2-positive.

319

Similar to other GPCRs [26], GnRHR forms functionally active homomers and heteromers with320

different receptors [7, 27]. We obtained a strong positive overlap coefficient between ACE2 and321

GnRHR on the cell surface of bovine gonadotrophs. Therefore, ACE2 may form a heteromer with

322

GnRHR in gonadotrophs.

323

However, the results of this study must be interpreted with some caution. Gu et al. [8]324reported ACE2 expression in the human AP gland, but found no significant difference in blood LH and325

FSH concentrations between and SARS-CoV2-infected and uninfected patients. However, in another326

study, male patients with COVID-19 showed lower blood concentrations of testosterone and higher

327

blood concentrations of LH and prolactin [28]. ACE2 is expressed at higher levels in the human testis328

and ovary than in the AP gland [29]. Therefore, one possible reason is that gonadal ACE2 may bind329

spike proteins rather than ACE2, and the direct and indirect suppression of steroidogenesis by the virus

330

and immune system, respectively, may then induce hypogonadism, which reduces the negative

331

feedback of steroid hormones to gonadotrophs [30]. Another possible reason could be that the332

measurements were performed on a one-point sample and not repeated to measure the333parameter of pulsatile secretion of LH and FSH.

334

Bovine ACE2 can bind the spike protein of SARS-CoV-2 [14] and SARS-CoV-2 replicates in335

bovine respiratory tissues [15]. Intratracheal and intravenous inoculation with SARS-CoV2 resulted in336

only minor replication in colostrum-deprived Holstein bull calves [31]. However, white-tailed deer

337

(Odocoileus virginianus), a ruminant species, are highly susceptible to infection [32]. Therefore,338

caution is needed against the spillover of SARS-CoV-2, especially of new variants, among humans,

339

domestic animals, and wild animals.

340

The primary role of ACE2 is to convert angiotensin II to angiotensin-(1–7). Although341

gonadotrophs are the likely site of angiotensin II production in rat AP glands [33], little is known

342

about the roles of ACE2, angiotensin II, and angiotensin-(1–7) in gonadotrophs. Therefore, further

343

studies are required to clarify these roles.

344

There are no previous reports of bovine ACE2 protein size using western blotting. As

345determined by western blotting, the bovine ACE2 protein was approximately 100 kDa. . However, this346

was larger than the 91 kDa predicted from the amino acid sequence. Additionally, human ACE2 shows347

a band at approximately 100 kDa along with a 50-kDa band [34]. In a previous study [19], HepG2

348

cells showed an approximately 100-kDa band using the same anti-ACE2 antibody, but showed a349

50-kDa band in this study. This difference in band size may be due to experimental conditions in the350

culture or sample preparation for western blot. This is because band sizes in western blotting often

351

differ from expected sizes in the case of membrane-bound proteins such as cell-surface receptors,

352

owing to their complex three-dimensional structures, which can include hydrophilic and lipophilic353

regions that form extracellular, transmembrane, and cytoplasmic domains [35].

354

Primary AP cell culture was used to evaluate rapidly activated (within 30 min)355

pathways that altered LH and FSH secretion from bovine gonadotrophs, without alterations in356

the mRNA expression of LHα, LHβ, or FSHβ subunits. Thus, we did not perform RT-qPCR

357

for LH and FSH genes. We previously found that the small interfering RNA method could not358

be used in this system because secretion was suppressed even in control RNA (unpublished359data). Therefore, we evaluated the contribution of ERK pathway using an inhibitor. We could360

not exclude any other pathway as U0126 partially recovered gonadotropin secretion. In

361

particular, Smad7 pathway suppresses cell function downstream of ACE2 in diabetic

362

nephropathy [36], and inhibits FSHB gene expression in mouse gonadotrope-derived LβT2

363

cells [37]. However, to the best of our knowledge, a SMAD7-specific inhibitor has not yet

364

been developed. Therefore, further studies are required in the future to investigate this aspect.

365

Angiotensin II activates the type 1 receptor (AT1R) and ERK pathways in rat tubular

366

epithelial cells [38]. Thus, if ACE2 is inhibited by the spike protein, angiotensin II may be increased367

and stimulate the AT1R and ERK pathways. However, to the best of our knowledge, there are no368

reports on the expression of AT1R in gonadotrophs. In addition, the culture medium did not contain369

angiotensin II. Therefore, it is unlikely that the angiotensin II-AT1R system contributed to the370

observed suppression by spike protein.

371

Moreover, non-gonadotroph cells were also ACE2-positive. Mice that ubiquitously overexpress

372

ACE2 have reduced the expression of proopiomelanocortin and plasma corticosterone in the AP [39].

373

ACE2 activation by diminazene aceturate increases ACTH secretion from AtT-20 cells but not374

prolactin secretion from MMQ and GH3 cells [8]. Therefore, ACE2-positive non-gonadotroph cells

375

may be corticotrophs.

376

In conclusion, ACE2 is expressed in gonadotrophs, and the SARS-CoV-2 spike protein

377

significantly suppresses LH and FSH secretion via the ERK pathway. This effect may constitute a378

cause of infertility in cows; however, further long-term in vivo studies are required to validate these379

results.

380
381

Conflict of Interest

382

The authors declare no conflicts of interest.

383
384

Acknowledgments

385

The authors thank Prof. Vitaliano Borromeo (Università degli Studi di Milano) for the anti-FSH

386

antibody. The authors thank Dr. A. F. Parlow (National Hormone and Peptide Program, Harbor-UCLA

387

Medical Center, Torrance, CA, USA) for hormones and antiserum. This research was partly supported

388

by a Grant-in-Aid for Scientific Research from The Research Institute for Time Studies, Yamaguchi

389

University (Yamaguchi, Japan) to H. Kadokawa.

390
391

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converting

enzyme2

dampensstressresponsiveness

in

male

mice.

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Figure Legends

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Fig. 1. Expression of Angiotensin-converting enzyme 2 (ACE2) detected using RT-PCR and western

507

blotting. Electrophoresis of PCR-amplified DNA products using primers for bovine ACE2 and cDNA

508

from bovine anterior pituitary (AP) glands; the band was 470 bp (A). Two bands (100 kDa and 42509kDa) appeared on the AP sample, whereas a 50-kDa band was observed in HepG2 cells, which were510

used as the positive control (B). The relative band of -actin (41 kDa) was used as a control for both511

HepG2 and AP (C).

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Fig. 2. Triple-fluorescence immunohistochemistry of bovine AP tissue for ACE2,

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gonadotropin-releasing hormone receptor (GnRHR), and either luteinizing hormone (LH) (A) or

534

follicle stimulating hormone (FSH) (B). Images were captured using laser confocal microscopy for LH

535

or FSH (red), ACE2 (green), and GnRHR (light blue) with counter-staining using DAPI (dark blue).

536

The yellow arrows indicate the colocalisation of ACE2 with GnRHR. Scale bars are 20 m.

537

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Fig. 3. Triple-fluorescence immunocytochemistry of cultured AP cells (prepared using PFA-Triton

542

method) of post-pubertal heifers for ACE2, GnRHR, and either LH (A) or FSH (B). Images were543

captured using laser confocal microscopy for LH or FSH (red), ACE2 (green), and GnRHR (light544blue) with counter-staining using DAPI (dark blue). The yellow arrows indicate the colocalisation of545

ACE2 with GnRHR. Scale bars are 20 m.

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Fig. 4. Fluorescence immunocytochemistry was used to confirm the colocalisation (yellow in the551

merge panel) of ACE2 and GnRHR on the surface of cultured AP cells (prepared using the Cell Cover552

method) of post-pubertal heifers. Images were captured using a laser confocal microscope for GnRHR

553(red), ACE2 (green), DNA (dark blue), and differential interference contrast (DIC) on cultured AP

554

cells which did not receive Triton X-100 treatment for antibody penetration. Thus, the antibody could

555

only bind ACE2 and GnRHR on the surface of gonadotrophs. The arrows indicate the colocalisation of556

ACE2 with GnRHR. Note that cells prepared using the Cell Cover method are thicker than those557

prepared using the PFA-Triton method. Scale bars are 10 m.

558

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Fig. 5. Comparison of the effects of various concentrations of spike protein in media without (A, C)

561

and with (B, D) 1 nM GnRH on LH or FSH secretion from cultured AP cells of post-pubertal heifers.

562

All cultured cells were pre-treated with activin. The concentrations of LH or FSH in control cells

563(cultured in medium alone without spike protein and GnRH) were averaged and set at 100%; the mean

564

LH or FSH concentration for each treatment group is expressed as a percentage of the control value.

565

Different letters indicate statistically significant differences (P < 0.05).

566

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Fig. 6. Effect of the ERK pathway inhibitor, U0126, on spike protein-mediated suppression of580

GnRH-induced secretion of LH (A) and FSH (B) from cultured bovine AP cells. All cultured cells

581

were pre-treated with activin. The mean LH or FSH concentration for each treatment group is582

expressed as a percentage of the control value. Different letters indicate statistically significant

583

differences (P < 0.05).

584

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Supplementary Fig. 1. Alignment of human (BAB40370) and bovine ACE2 (NP_001019673.2)

603

amino acid sequences using the basic local alignment search tool available on the NCBI website. Red

604

fonts indicate the key amino acids for severe acute respiratory syndrome coronavirus-2 binding

605

reported by Luan et al. [14]. Green fonts indicate N-linked glycosylation sites. Pink fonts indicate the606

phosphorylation sites according to the annotation of each protein. The grey region indicates the signal607

peptide. The green region indicates the antigen peptide immunized to create the used anti-human

608

ACE2 antibody, where the homology between human and bovine ACE2 is 86% (79/92) and gap is 0%609(79/92). The orange region indicates the target of RT-PCR. The blue region indicates the610transmembrane region.